Mutations in CAPN1 Cause Autosomal-Recessive Hereditary Spastic Paraplegia

Hereditary spastic paraplegia (HSP) is a genetically and clinically heterogeneous disease characterized by spasticity and weakness of the lower limbs with or without additional neurological symptoms. Although more than 70 genes and genetic loci have been implicated in HSP, many families remain genetically undiagnosed, suggesting that other genetic causes of HSP are still to be identified. HSP can be inherited in an autosomal-dominant, autosomal-recessive, or X-linked manner. In the current study, we performed whole-exome sequencing to analyze a total of nine affected individuals in three families with autosomal-recessive HSP. Rare homozygous and compound-heterozygous nonsense, missense, frameshift, and splice-site mutations in CAPN1 were identified in all affected individuals, and sequencing in additional family members confirmed the segregation of these mutations with the disease (spastic paraplegia 76 [SPG76]). CAPN1 encodes calpain 1, a protease that is widely present in the CNS. Calpain 1 is involved in synaptic plasticity, synaptic restructuring, and axon maturation and maintenance. Three models of calpain 1 deficiency were further studied. In Caenorhabditis elegans, loss of calpain 1 function resulted in neuronal and axonal dysfunction and degeneration. Similarly, loss-of-function of the Drosophila melanogaster ortholog calpain B caused locomotor defects and axonal anomalies. Knockdown of calpain 1a, a CAPN1 ortholog in Danio rerio, resulted in abnormal branchiomotor neuron migration and disorganized acetylated-tubulin axonal networks in the brain. The identification of mutations in CAPN1 in HSP expands our understanding of the disease causes and potential mechanisms.     

Mutations in BICD2 Cause Dominant Congenital Spinal Muscular Atrophy and Hereditary Spastic Paraplegia

Dominant congenital spinal muscular atrophy (DCSMA) is a disorder of developing anterior horn cells and shows lower-limb predominance and clinical overlap with hereditary spastic paraplegia (HSP), a lower-limb-predominant disorder of corticospinal motor neurons. We have identified four mutations in bicaudal D homolog 2 (Drosophila) (BICD2) in six kindreds affected by DCSMA, DCSMA with upper motor neuron features, or HSP. BICD2 encodes BICD2, a key adaptor protein that interacts with the dynein-dynactin motor complex, which facilitates trafficking of cellular cargos that are critical to motor neuron development and maintenance. We demonstrate that mutations resulting in amino acid substitutions in two binding regions of BICD2 increase its binding affinity for the cytoplasmic dynein-dynactin complex, which might result in the perturbation of BICD2-dynein-dynactin-mediated trafficking, and impair neurite outgrowth. These findings provide insight into the mechanism underlying both the static and the slowly progressive clinical features and the motor neuron pathology that characterize BICD2-associated diseases, and underscore the importance of the dynein-dynactin transport pathway in the development and survival of both lower and upper motor neurons.     

Alteration of Fatty-Acid-Metabolizing Enzymes Affects Mitochondrial Form and Function in Hereditary Spastic Paraplegia

Hereditary spastic paraplegia (HSP) is considered one of the most heterogeneous groups of neurological disorders, both clinically and genetically. The disease comprises pure and complex forms that clinically include slowly progressive lower-limb spasticity resulting from degeneration of the corticospinal tract. At least 48 loci accounting for these diseases have been mapped to date, and mutations have been identified in 22 genes, most of which play a role in intracellular trafficking. Here, we identified mutations in two functionally related genes (DDHD1 and CYP2U1) in individuals with autosomal-recessive forms of HSP by using either the classical positional cloning or a combination of whole-genome linkage mapping and next-generation sequencing. Interestingly, three subjects with CYP2U1 mutations presented with a thin corpus callosum, white-matter abnormalities, and/or calcification of the basal ganglia. These genes code for two enzymes involved in fatty-acid metabolism, and we have demonstrated in human cells that the HSP pathophysiology includes alteration of mitochondrial architecture and bioenergetics with increased oxidative stress. Our combined results focus attention on lipid metabolism as a critical HSP pathway with a deleterious impact on mitochondrial bioenergetic function.     

A Hereditary Enteropathy Caused by Mutations in the SLCO2A1 Gene,Encoding a Prostaglandin Transporter

Previously, we proposed a rare autosomal recessive inherited enteropathy characterized by persistent blood and protein loss from the small intestine as chronic nonspecific multiple ulcers of the small intestine (CNSU). By whole-exome sequencing in five Japanese patients with CNSU and one unaffected individual, we found four candidate mutations in the SLCO2A1 gene, encoding a prostaglandin transporter. The pathogenicity of the mutations was supported by segregation analysis and genotyping data in controls. By Sanger sequencing of the coding regions, 11 of 12 other CNSU patients and 2 of 603 patients with a diagnosis of Crohn’s disease were found to have homozygous or compound heterozygous SLCO2A1 mutations. In total, we identified recessive SLCO2A1 mutations located at seven sites. Using RT-PCR, we demonstrated that the identified splice-site mutations altered the RNA splicing, and introduced a premature stop codon. Tracer prostaglandin E2 uptake analysis showed that the mutant SLCO2A1 protein for each mutation exhibited impaired prostaglandin transport. Immunohistochemistry and immunofluorescence analyses revealed that SLCO2A1 protein was expressed on the cellular membrane of vascular endothelial cells in the small intestinal mucosa in control subjects, but was not detected in affected individuals. These findings indicate that loss-of-function mutations in the SLCO2A1 gene encoding a prostaglandin transporter cause the hereditary enteropathy CNSU. We suggest a more appropriate nomenclature of “chronic enteropathy associated with SLCO2A1 gene” (CEAS).     

Mutations in CYC1, Encoding Cytochrome c1 Subunit of Respiratory Chain Complex III,Cause Insulin-Responsive Hyperglycemia

Many individuals with abnormalities of mitochondrial respiratory chain complex III remain genetically undefined. Here, we report mutations (c.288G>T [p.Trp96Cys] and c.643C>T [p.Leu215Phe]) in CYC1, encoding the cytochrome c₁ subunit of complex III, in two unrelated children presenting with recurrent episodes of ketoacidosis and insulin-responsive hyperglycemia. Cytochrome c₁, the heme-containing component of complex III, mediates the transfer of electrons from the Rieske iron-sulfur protein to cytochrome c. Cytochrome c₁ is present at reduced levels in the skeletal muscle and skin fibroblasts of affected individuals. Moreover, studies on yeast mutants and affected individuals’ fibroblasts have shown that exogenous expression of wild-type CYC1 rescues complex III activity, demonstrating the deleterious effect of each mutation on cytochrome c₁ stability and complex III activity.     

Recessive Mutations in KCNJ13, Encoding an Inwardly Rectifying Potassium Channel Subunit, Cause Leber Congenital Amaurosis

Inherited retinal degenerations, including retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), comprise a group of disorders showing high genetic and allelic heterogeneity. The determination of a full catalog of genes that can, when mutated, cause human retinal disease is a powerful means to understand the molecular physiology and pathology of the human retina. As more genes are found, remaining ones are likely to be rarer and/or unexpected candidates. Here, we identify a family in which all known RP/LCA-related genes are unlikely to be associated with their disorder. A combination of homozygosity mapping and exome sequencing identifies a homozygous nonsense mutation, c.496C>T (p.Arg166X), in a gene, KCNJ13, encoding a potassium channel subunit Kir7.1. A screen of a further 333 unrelated individuals with recessive retinal degeneration identified an additional proband, homozygous for a missense mutation, c.722T>C (p.Leu241Pro), in the same gene. The three affected members of the two families have been diagnosed with LCA. All have a distinct and unusual retinal appearance and a similar early onset of visual loss, suggesting both impaired retinal development and progressive retinal degeneration, involving both rod and cone pathways. Examination of heterozygotes revealed no ocular disease. This finding implicates Kir7.1 as having an important role in human retinal development and maintenance. This disorder adds to a small diverse group of diseases consequent upon loss or reduced function of inwardly rectifying potassium channels affecting various organs. The distinct retinal phenotype that results from biallelic mutations in KCNJ13 should facilitate the molecular diagnosis in further families.     

Recessive Mutations in COL25A1 Are a Cause of Congenital Cranial Dysinnervation Disorder

Abnormal ocular motility is a common clinical feature in congenital cranial dysinnervation disorder (CCDD). To date, eight genes related to neuronal development have been associated with different CCDD phenotypes. By using linkage analysis, candidate gene screening, and exome sequencing, we identified three mutations in collagen, type XXV, alpha 1 (COL25A1) in individuals with autosomal-recessive inheritance of CCDD ophthalmic phenotypes. These mutations affected either stability or levels of the protein. We further detected altered levels of sAPP (neuronal protein involved in axon guidance and synaptogenesis) and TUBB3 (encoded by TUBB3, which is mutated in CFEOM3) as a result of null mutations in COL25A1. Our data suggest that lack of COL25A1 might interfere with molecular pathways involved in oculomotor neuron development, leading to CCDD phenotypes.     

Mutations in POGLUT1, Encoding Protein O-Glucosyltransferase 1, Cause Autosomal-Dominant Dowling-Degos Disease

Dowling-Degos disease (DDD) is an autosomal-dominant genodermatosis characterized by progressive and disfiguring reticulate hyperpigmentation. We previously identified loss-of-function mutations in KRT5 but were only able to detect pathogenic mutations in fewer than half of our subjects. To identify additional causes of DDD, we performed exome sequencing in five unrelated affected individuals without mutations in KRT5. Data analysis identified three heterozygous mutations from these individuals, all within the same gene. These mutations, namely c.11G>A (p.Trp4^∗), c.652C>T (p.Arg218^∗), and c.798-2A>C, are within POGLUT1, which encodes protein O-glucosyltransferase 1. Further screening of unexplained cases for POGLUT1 identified six additional mutations, as well as two of the above described mutations. Immunohistochemistry of skin biopsies of affected individuals with POGLUT1 mutations showed significantly weaker POGLUT1 staining in comparison to healthy controls with strong localization of POGLUT1 in the upper parts of the epidermis. Immunoblot analysis revealed that translation of either wild-type (WT) POGLUT1 or of the protein carrying the p.Arg279Trp substitution led to the expected size of about 50 kDa, whereas the c.652C>T (p.Arg218^∗) mutation led to translation of a truncated protein of about 30 kDa. Immunofluorescence analysis identified a colocalization of the WT protein with the endoplasmic reticulum and a notable aggregating pattern for the truncated protein. Recently, mutations in POFUT1, which encodes protein O-fucosyltransferase 1, were also reported to be responsible for DDD. Interestingly, both POGLUT1 and POFUT1 are essential regulators of Notch activity. Our results furthermore emphasize the important role of the Notch pathway in pigmentation and keratinocyte morphology.     

Mutations in POFUT1, Encoding Protein O-fucosyltransferase 1, Cause Generalized Dowling-Degos Disease

Dowling-Degos disease (DDD), or reticular pigmented anomaly of the flexures, is a type of rare autosomal-dominant genodermatosis characterized by reticular hyperpigmentation and hypopigmentation of the flexures, such as the neck, axilla, and areas below the breasts and groin, and shows considerable heterogeneity. Loss-of-function mutations of keratin 5 (KRT5) have been identified in DDD individuals. In this study, we collected DNA samples from a large Chinese family affected by generalized DDD and found no mutation of KRT5. We performed a genome-wide linkage analysis of this family and mapped generalized DDD to a region between rs1293713 and rs244123 on chromosome 20. By exome sequencing, we identified nonsense mutation c.430G>T (p.Glu144^∗) in POFUT1, which encodes protein O-fucosyltransferase 1, in the family. Study of an additional generalized DDD individual revealed the heterozygous deletion mutation c.482delA (p.Lys161Serfs^∗42) in POFUT1. Knockdown of POFUT1 reduces the expression of NOTCH1, NOTCH2, HES1, and KRT5 in HaCaT cells. Using zebrafish, we showed that pofut1 is expressed in the skin and other organs. Morpholino knockdown of pofut1 in zebrafish produced a phenotype characteristic of hypopigmentation at 48 hr postfertilization (hpf) and abnormal melanin distribution at 72 hpf, replicating the clinical phenotype observed in our DDD individuals. At 48 and 72 hpf, tyrosinase activities decreased by 33% and 45%, respectively, and melanin protein contents decreased by 20% and 25%, respectively. Our findings demonstrate that POFUT1 mutations cause generalized DDD. These results strongly suggest that the protein product of POFUT1 plays a significant and conserved role in melanin synthesis and transport.     

Mutations in SEC24D,Encoding a Component of the COPII Machinery,Cause a Syndromic Form of Osteogenesis Imperfecta

As a result of a whole-exome sequencing study, we report three mutant alleles in SEC24D, a gene encoding a component of the COPII complex involved in protein export from the ER: the truncating mutation c.613C>T (p.Gln205^∗) and the missense mutations c.3044C>T (p.Ser1015Phe, located in a cargo-binding pocket) and c.2933A>C (p.Gln978Pro, located in the gelsolin-like domain). Three individuals from two families affected by a similar skeletal phenotype were each compound heterozygous for two of these mutant alleles, with c.3044C>T being embedded in a 14 Mb founder haplotype shared by all three. The affected individuals were a 7-year-old boy with a phenotype most closely resembling Cole-Carpenter syndrome and two fetuses initially suspected to have a severe type of osteogenesis imperfecta. All three displayed a severely disturbed ossification of the skull and multiple fractures with prenatal onset. The 7-year-old boy had short stature and craniofacial malformations including macrocephaly, midface hypoplasia, micrognathia, frontal bossing, and down-slanting palpebral fissures. Electron and immunofluorescence microscopy of skin fibroblasts of this individual revealed that ER export of procollagen was inefficient and that ER tubules were dilated, faithfully reproducing the cellular phenotype of individuals with cranio-lentico-sutural dysplasia (CLSD). CLSD is caused by SEC23A mutations and displays a largely overlapping craniofacial phenotype, but it is not characterized by generalized bone fragility and presented with cataracts in the original family described. The cellular and morphological phenotypes we report are in concordance with the phenotypes described for the Sec24d-deficient fish mutants vbi (medaka) and bulldog (zebrafish).     

Loss of Function of Glucocerebrosidase GBA2 Is Responsible for Motor Neuron Defects in Hereditary Spastic Paraplegia

Spastic paraplegia 46 refers to a locus mapped to chromosome 9 that accounts for a complicated autosomal-recessive form of hereditary spastic paraplegia (HSP). With next-generation sequencing in three independent families, we identified four different mutations in GBA2 (three truncating variants and one missense variant), which were found to cosegregate with the disease and were absent in controls. GBA2 encodes a microsomal nonlysosomal glucosylceramidase that catalyzes the conversion of glucosylceramide to free glucose and ceramide and the hydrolysis of bile acid 3-O-glucosides. The missense variant was also found at the homozygous state in a simplex subject in whom no residual glucocerebrosidase activity of GBA2 could be evidenced in blood cells, opening the way to a possible measurement of this enzyme activity in clinical practice. The overall phenotype was a complex HSP with mental impairment, cataract, and hypogonadism in males associated with various degrees of corpus callosum and cerebellar atrophy on brain imaging. Antisense morpholino oligonucleotides targeting the zebrafish GBA2 orthologous gene led to abnormal motor behavior and axonal shortening/branching of motoneurons that were rescued by the human wild-type mRNA but not by applying the same mRNA containing the missense mutation. This study highlights the role of ceramide metabolism in HSP pathology.