Cathepsin A-related arteriopathy with strokes and leukoencephalopathy (CARASAL).

OBJECTIVE: To characterize the clinical and MRI features of 2 families with adult-onset dominant leukoencephalopathy and strokes and identify the underlying genetic cause. METHODS: We applied MRI pattern recognition, whole-exome sequencing, and neuropathology. RESULTS: Based on brain imaging, 13 family members of 40 years or older from 2 families were diagnosed with the disease; in 11 family members of the same age, MRI was normal. In the affected family members, MRI showed a leukoencephalopathy that was disproportionately severe compared to the clinical disease. The clinical picture was dominated by ischemic and hemorrhagic strokes, slow and late cognitive deterioration, and therapy-resistant hypertension. With whole-exome sequencing, we identified one variant shared by both families and segregating with the disease: c.973C>T in CTSA. Haplotype analysis revealed a shared 1,145-kb interval encompassing the CTSA variant on chromosome 20q13.12, suggesting a common ancestor. Brain autopsy of 3 patients showed a leukoencephalopathy that was disproportionately extensive compared to the vascular abnormalities. CTSA encodes cathepsin A. Recessive CTSA mutations cause galactosialidosis. One of the numerous cathepsin A functions is to degrade endothelin-1. In the patients, striking endothelin-1 immunoreactivity was found in white matter astrocytes, correlating with increased numbers of premyelinating oligodendrocyte progenitors. This finding supports a role for endothelin-1 in the leukoencephalopathy through inhibition of oligodendrocyte progenitor maturation. CONCLUSIONS: CARASAL (cathepsin A-related arteriopathy with strokes and leukoencephalopathy) is a novel hereditary adult-onset cerebral small vessel disease. It is of interest that, next to the cerebral vascular abnormalities, endothelin-1 may have a role in the pathogenesis of the extensive leukoencephalopathy.

Update on Leukodystrophies: A Historical Perspective and Adapted Definition.

Leukodystrophies were defined in the 1980s as progressive genetic disorders primarily affecting myelin of the central nervous system. At that time, a limited number of such disorders and no associated gene defects were known. The majority of the leukodystrophy patients remained without a specific diagnosis. In the following two decades, magnetic resonance imaging pattern recognition revolutionized the field, allowing the definition of numerous novel leukodystrophies. Their genetic defects were usually identified through genetic linkage studies. This process required substantial numbers of cases and many rare disorders remained unclarified. As recently as 2010, 50% of the leukodystrophy patients remained unclassified. Since 2011, whole-exome sequencing has resulted in an exponential increase in numbers of known, distinct, genetically determined, ultrarare leukodystrophies. We performed a retrospective study concerning three historical cohorts of unclassified leukodystrophy patients and found that currently at least 80% of the patients can be molecularly classified. Based on the original definition of the leukodystrophies, numerous defects in proteins important in myelin structure, maintenance, and function were expected. By contrast, a high percentage of the newly identified gene defects affect the housekeeping process of mRNA translation, shedding new light on white matter pathobiology and requiring adaptation of the leukodystrophy definition.

Acute intermittent porphyria-related leukoencephalopathy.

OBJECTIVE: To identify the genetic etiology of a distinct leukoencephalopathy with autosomal recessive inheritance in a single family. METHODS: We analyzed available MRIs and retrospectively reviewed clinical information and laboratory investigations. We performed whole-exome sequencing to find the causal gene variants. RESULTS: We identified 3 family members with a similar MRI pattern characterized by symmetrical signal abnormalities in the periventricular and deep cerebral white matter, thalami, and central part of the pons. Cerebellar atrophy was noted in advanced disease stages. Clinical features were childhood-onset slowly progressive spastic paraparesis, cerebellar ataxia, peripheral neuropathy, and in 2 patients, optic atrophy as well as vertical gaze and convergence palsies and nystagmus. Whole-exome sequencing revealed compound heterozygous missense variants in the HMBS gene, both associated with the autosomal dominant disorder acute intermittent porphyria. Sanger sequencing of 6 healthy siblings confirmed the bi-allelic location of the variants and segregation with the disease. Patients had a slight and moderate increase in urinary and plasma porphobilinogen and 5'-aminolevulinic acid, respectively, and a 50% to 66% decrease in hydroxymethylbilane synthase enzyme activity compared to normal. CONCLUSIONS: Bi-allelic HMBS variants have been reported before as cause of severe encephalopathy with early childhood fatality in acute intermittent porphyria. Our cases demonstrate childhood onset, but milder and slower disease progression in middle-aged patients. With this, a novel phenotype can be added to the disease spectrum associated with bi-allelic HMBS variants: a leukoencephalopathy with early onset, slowly progressive neurologic symptomatology, and long life expectancy.

Absent Thalami Caused by a Homozygous EARS2 Mutation: Expanding Disease Spectrum of LTBL.

Leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL) is caused by autosomal recessive EARS2 mutations. Onset is most often in infancy, but in severe cases in the neonatal period. Patients typically have magnetic resonance imaging (MRI) signal abnormalities involving the thalamus, brainstem, and deep cerebral white matter. Most signal abnormalities resolve, but in severe cases at the expense of tissue loss. Here, we report a patient with an encephalopathy of antenatal onset. His early MRI at 8 months of age showed signal abnormalities in the deep cerebral white matter that improved over time. The thalami were absent with the configuration of a developmental anomaly, without evidence of a lesion. We hypothesized that this was a case of LTBL in which the thalamic damage occurred antenatally and was incorporated in the normal brain development. The diagnosis was confirmed by a novel homozygous EARS2 mutation. Our case adds to the phenotypic and genetic spectrum of LTBL.

Early-Onset Severe Encephalopathy with Epilepsy: The BRAT1 Gene Should Be Added to the List of Causes.

A variety of pathologies can underlie early-onset severe encephalopathy with epilepsy. To aid the diagnostic process in such patients we present an overview of causes, including the rapidly expanding list of genes involved. When no explanation is found, whole-exome sequencing (WES) can be used in an attempt to identify gene defects in patients suspected to suffer from a genetic form. We describe three siblings, born to consanguineous parents, with a lethal severe epileptic encephalopathy with early-infantile onset, including their magnetic resonance imaging, electroencephalography and, in one case, neuropathological findings. Using WES a homozygous frameshift mutation in the BRAT1 gene, c.638dup p.(Val214Glyfs*189), was identified. We present our cases in the context of all published cases with mutations in the BRAT1 gene and conclude that BRAT1 should be added to the growing list of genes related to early-onset severe encephalopathy with epilepsy.

Altered PLP1 splicing causes hypomyelination of early myelinating structures.

OBJECTIVE: The objective of this study was to investigate the genetic etiology of the X-linked disorder "Hypomyelination of Early Myelinating Structures" (HEMS). METHODS: We included 16 patients from 10 families diagnosed with HEMS by brain MRI criteria. Exome sequencing was used to search for causal mutations. In silico analysis of effects of the mutations on splicing and RNA folding was performed. In vitro gene splicing was examined in RNA from patients' fibroblasts and an immortalized immature oligodendrocyte cell line after transfection with mutant minigene splicing constructs. RESULTS: All patients had unusual hemizygous mutations of PLP1 located in exon 3B (one deletion, one missense and two silent), which is spliced out in isoform DM20, or in intron 3 (five mutations). The deletion led to truncation of PLP1, but not DM20. Four mutations were predicted to affect PLP1/DM20 alternative splicing by creating exonic splicing silencer motifs or new splice donor sites or by affecting the local RNA structure of the PLP1 splice donor site. Four deep intronic mutations were predicted to destabilize a long-distance interaction structure in the secondary PLP1 RNA fragment involved in regulating PLP1/DM20 alternative splicing. Splicing studies in fibroblasts and transfected cells confirmed a decreased PLP1/DM20 ratio. INTERPRETATION: Brain structures that normally myelinate early are poorly myelinated in HEMS, while they are the best myelinated structures in Pelizaeus-Merzbacher disease, also caused by PLP1 alterations. Our data extend the phenotypic spectrum of PLP1-related disorders indicating that normal PLP1/DM20 alternative splicing is essential for early myelination and support the need to include intron 3 in diagnostic sequencing.

Recessive ITPA mutations cause an early infantile encephalopathy.

OBJECTIVE: To identify the etiology of a novel, heritable encephalopathy in a small group of patients. METHODS: Magnetic resonance imaging (MRI) pattern analysis was used to select patients with the same pattern. Homozygosity mapping and whole exome sequencing (WES) were performed to find the causal gene mutations. RESULTS: Seven patients from 4 families (2 consanguineous) were identified with a similar MRI pattern characterized by T2 signal abnormalities and diffusion restriction in the posterior limb of the internal capsule, often also optic radiation, brainstem tracts, and cerebellar white matter, in combination with delayed myelination and progressive brain atrophy. Patients presented with early infantile onset encephalopathy characterized by progressive microcephaly, seizures, variable cardiac defects, and early death. Metabolic testing was unrevealing. Single nucleotide polymorphism array revealed 1 overlapping homozygous region on chromosome 20 in the consanguineous families. In all patients, WES subsequently revealed recessive predicted loss of function mutations in ITPA, encoding inosine triphosphate pyrophosphatase (ITPase). ITPase activity in patients' erythrocytes and fibroblasts was severely reduced. INTERPRETATION: Until now ITPA variants have only been associated with adverse reactions to specific drugs. This is the first report associating ITPA mutations with a human disorder. ITPase is important in purine metabolism because it removes noncanonical nucleotides from the cellular nucleotide pool. Toxicity of accumulated noncanonical nucleotides, leading to neuronal apoptosis and interference with proteins normally using adenosine triphosphate/guanosine triphosphate, probably explains the disease. This study confirms that combining MRI pattern recognition to define small, homogeneous patient groups with WES is a powerful approach for providing a fast diagnosis in patients with an unclassified genetic encephalopathy.

Novel (ovario) leukodystrophy related to AARS2 mutations.

OBJECTIVES: The study was focused on leukoencephalopathies of unknown cause in order to define a novel, homogeneous phenotype suggestive of a common genetic defect, based on clinical and MRI findings, and to identify the causal genetic defect shared by patients with this phenotype. METHODS: Independent next-generation exome-sequencing studies were performed in 2 unrelated patients with a leukoencephalopathy. MRI findings in these patients were compared with available MRIs in a database of unclassified leukoencephalopathies; 11 patients with similar MRI abnormalities were selected. Clinical and MRI findings were investigated. RESULTS: Next-generation sequencing revealed compound heterozygous mutations in AARS2 encoding mitochondrial alanyl-tRNA synthetase in both patients. Functional studies in yeast confirmed the pathogenicity of the mutations in one patient. Sanger sequencing revealed AARS2 mutations in 4 of the 11 selected patients. The 6 patients with AARS2 mutations had childhood- to adulthood-onset signs of neurologic deterioration consisting of ataxia, spasticity, and cognitive decline with features of frontal lobe dysfunction. MRIs showed a leukoencephalopathy with striking involvement of left-right connections, descending tracts, and cerebellar atrophy. All female patients had ovarian failure. None of the patients had signs of a cardiomyopathy. CONCLUSIONS: Mutations in AARS2 have been found in a severe form of infantile cardiomyopathy in 2 families. We present 6 patients with a new phenotype caused by AARS2 mutations, characterized by leukoencephalopathy and, in female patients, ovarian failure, indicating that the phenotypic spectrum associated with AARS2 variants is much wider than previously reported.

NUBPL mutations in patients with complex I deficiency and a distinct MRI pattern.

OBJECTIVE: To identify the mutated gene in a group of patients with an unclassified heritable white matter disorder sharing the same, distinct MRI pattern. METHODS: We used MRI pattern recognition analysis to select a group of patients with a similar, characteristic MRI pattern. We performed whole-exome sequencing to identify the mutated gene. We examined patients' fibroblasts for biochemical consequences of the mutant protein. RESULTS: We identified 6 patients from 5 unrelated families with a similar MRI pattern showing predominant abnormalities of the cerebellar cortex, deep cerebral white matter, and corpus callosum. The 4 tested patients had a respiratory chain complex І deficiency. Exome sequencing revealed mutations in NUBPL, encoding an iron-sulfur cluster assembly factor for complex І, in all patients. Upon identification of the mutated gene, we analyzed the MRI of a previously published case with NUBPL mutations and found exactly the same pattern. A strongly decreased amount of NUBPL protein and fully assembled complex I was found in patients' fibroblasts. Analysis of the effect of mutated NUBPL on the assembly of the peripheral arm of complex I indicated that NUBPL is involved in assembly of iron-sulfur clusters early in the complex I assembly pathway. CONCLUSION: Our data show that NUBPL mutations are associated with a unique, consistent, and recognizable MRI pattern, which facilitates fast diagnosis and obviates the need for other tests, including assessment of mitochondrial complex activities in muscle or fibroblasts.

Exome sequencing reveals mutated SLC19A3 in patients with an early-infantile, lethal encephalopathy.

To accomplish a diagnosis in patients with a rare unclassified disorder is difficult. In this study, we used magnetic resonance imaging pattern recognition analysis to identify patients with the same novel heritable disorder. Whole-exome sequencing was performed to discover the mutated gene. We identified seven patients sharing a previously undescribed magnetic resonance imaging pattern, characterized by initial swelling with T2 hyperintensity of the basal nuclei, thalami, cerebral white matter and cortex, pons and midbrain, followed by rarefaction or cystic degeneration of the white matter and, eventually, by progressive cerebral, cerebellar and brainstem atrophy. All patients developed a severe encephalopathy with rapid deterioration of neurological functions a few weeks after birth, followed by respiratory failure and death. Lactate was elevated in body fluids and on magnetic resonance spectroscopy in most patients. Whole-exome sequencing in a single patient revealed two predicted pathogenic, heterozygous missense mutations in the SLC19A3 gene, encoding the second thiamine transporter. Additional predicted pathogenic mutations and deletions were detected by Sanger sequencing in all six other patients. Pathology of brain tissue of two patients demonstrated severe cerebral atrophy and microscopic brain lesions similar to Leigh's syndrome. Although the localization of SLC19A3 expression in brain was similar in the two investigated patients compared to age-matched control subjects, the intensity of the immunoreactivity was increased. Previously published patients with SLC19A3 mutations have a milder clinical phenotype, no laboratory evidence of mitochondrial dysfunction and more limited lesions on magnetic resonance imaging. In some, cerebral atrophy has been reported. The identification of this new, severe, lethal phenotype characterized by subtotal brain degeneration broadens the phenotypic spectrum of SLC19A3 mutations. Recognition of the associated magnetic resonance imaging pattern allows a fast diagnosis in affected infants.

Gain of glycosylation in integrin α3 causes lung disease and nephrotic syndrome.

Integrins are transmembrane αß glycoproteins that connect the extracellular matrix to the cytoskeleton. The laminin-binding integrin α3ß1 is expressed at high levels in lung epithelium and in kidney podocytes. In podocytes, α3ß1 associates with the tetraspanin CD151 to maintain a functional filtration barrier. Here, we report on a patient homozygous for a novel missense mutation in the human ITGA3 gene, causing fatal interstitial lung disease and congenital nephrotic syndrome. The mutation caused an alanine-to-serine substitution in the integrin α3 subunit, thereby introducing an N-glycosylation motif at amino acid position 349. We expressed this mutant form of ITGA3 in murine podocytes and found that hyperglycosylation of the α3 precursor prevented its heterodimerization with ß1, whereas CD151 association with the α3 subunit occurred normally. Consequently, the ß1 precursor accumulated in the ER, and the mutant α3 precursor was degraded by the ubiquitin-proteasome system. Thus, these findings uncover a gain-of-glycosylation mutation in ITGA3 that prevents the biosynthesis of functional α3ß1, causing a fatal multiorgan disorder.

Copy number variations in patients with electrical status epilepticus in sleep.

Electrical status epilepticus in sleep syndrome is the association of the electroencephalographic pattern and deficits in language or global cognitive function and behavioral problems. The etiology is often unknown, but genetic risk factors have been implicated. Array-based comparative genomic hybridization was used to identify copy number variations in 13 children with electrical status epilepticus in sleep syndrome to identify possible underlying risk factors. Seven copy number variations were detected in 4 of the 13 patients, which consisted of 6 novel gains and 1 loss, the recurrent 15q13.3 microdeletion. Two patients carried a probable pathogenic copy number variation containing a gene involved in the cholinergic pathway. Genetic aberrations in patients with electrical status epilepticus in sleep syndrome can provide an entry in the investigation of the etiology of electrical status epilepticus in sleep. However, further studies are needed to confirm our findings.

A patient with a mild holoprosencephaly spectrum phenotype and heterotaxy and a 1.3 Mb deletion encompassing GLI2.

Loss-of-function mutations of GLI2 are associated with features at the mild end of the holoprosencephaly spectrum, including abnormal pituitary gland formation and/or function, and craniofacial abnormalities. In addition patients may have branchial arch anomalies and polydactyly. Large, microscopically visible, interstitial deletions spanning 2q14.2 have been reported in patients with multiple congenital anomalies and intellectual disability. We report here on a patient with a mild holoprosencephaly spectrum phenotype (bilateral cleft lip and palate and abnormal pituitary gland formation with panhypopituitarism) and normal psychomotor development, who was found to carry a 1.3 Mb submicroscopic heterozygous deletion in 2q14.2, encompassing the GLI2 gene. We review the genotype and phenotype of previously published probands with GLI2 aberrations. Our findings confirm the association of haploinsufficiency of GLI2 and mild HPE spectrum features. Consistent with prior reports, we observed incomplete penetrance of the deletion in the family, illustrating the multifactorial etiology of holoprosencephaly spectrum features. In addition to the holoprosencephaly spectrum features, the proband had heterotaxy of the abdominal organs. Mutations in the known heterotaxy genes (NODAL, ZIC3 and CFC1) were excluded. The deletion contains five genes, in addition to GLI2, including the EPB4.1l5 gene. Based on findings in Epb4.1l5 mutant mice we hypothesize that Epb4.1l5 is a candidate gene for the heterotaxy observed in the proband.