Deep Intronic FGF14 GAA Repeat Expansion in Late-Onset Cerebellar Ataxia.

BACKGROUND: The late-onset cerebellar ataxias (LOCAs) have largely resisted molecular diagnosis. METHODS: We sequenced the genomes of six persons with autosomal dominant LOCA who were members of three French Canadian families and identified a candidate pathogenic repeat expansion. We then tested for association between the repeat expansion and disease in two independent case-control series - one French Canadian (66 patients and 209 controls) and the other German (228 patients and 199 controls). We also genotyped the repeat in 20 Australian and 31 Indian index patients. We assayed gene and protein expression in two postmortem cerebellum specimens and two induced pluripotent stem-cell (iPSC)-derived motor-neuron cell lines. RESULTS: In the six French Canadian patients, we identified a GAA repeat expansion deep in the first intron of FGF14, which encodes fibroblast growth factor 14. Cosegregation of the repeat expansion with disease in the families supported a pathogenic threshold of at least 250 GAA repeats ([GAA]≥250). There was significant association between FGF14 (GAA)≥250 expansions and LOCA in the French Canadian series (odds ratio, 105.60; 95% confidence interval [CI], 31.09 to 334.20; P<0.001) and in the German series (odds ratio, 8.76; 95% CI, 3.45 to 20.84; P<0.001). The repeat expansion was present in 61%, 18%, 15%, and 10% of French Canadian, German, Australian, and Indian index patients, respectively. In total, we identified 128 patients with LOCA who carried an FGF14 (GAA)≥250 expansion. Postmortem cerebellum specimens and iPSC-derived motor neurons from patients showed reduced expression of FGF14 RNA and protein. CONCLUSIONS: A dominantly inherited deep intronic GAA repeat expansion in FGF14 was found to be associated with LOCA. (Funded by Fondation Groupe Monaco and others.).

Partial loss-of-function variant in neuregulin 1 identified in family with heritable peripheral neuropathy.

Neuregulin 1 signals are essential for the development and function of Schwann cells, which form the myelin sheath on peripheral axons. Disruption of myelin in the peripheral nervous system can lead to peripheral neuropathy, which is characterized by reduced axonal conduction velocity and sensorimotor deficits. Charcot-Marie-Tooth disease is a group of heritable peripheral neuropathies that may be caused by variants in nearly 100 genes. Despite the evidence that Neuregulin 1 is essential for many aspects of Schwann cell development, previous studies have not reported variants in the neuregulin 1 gene (NRG1) in patients with peripheral neuropathy. We have identified a rare missense variant in NRG1 that is homozygous in a patient with sensory and motor deficits consistent with mixed axonal and de-myelinating peripheral neuropathy. Our in vivo functional studies in zebrafish indicate that the patient variant partially reduces NRG1 function. This study tentatively suggests that variants at the NRG1 locus may cause peripheral neuropathy and that NRG1 should be investigated in families with peripheral neuropathy of unknown cause.

Neurogenetic fetal akinesia and arthrogryposis: genetics, expanding genotype-phenotypes and functional genomics.

BACKGROUND: Fetal akinesia and arthrogryposis are clinically and genetically heterogeneous and have traditionally been refractive to genetic diagnosis. The widespread availability of affordable genome-wide sequencing has facilitated accurate genetic diagnosis and gene discovery in these conditions. METHODS: We performed next generation sequencing (NGS) in 190 probands with a diagnosis of arthrogryposis multiplex congenita, distal arthrogryposis, fetal akinesia deformation sequence or multiple pterygium syndrome. This sequencing was a combination of bespoke neurogenetic disease gene panels and whole exome sequencing. Only class 4 and 5 variants were reported, except for two cases where the identified variants of unknown significance (VUS) are most likely to be causative for the observed phenotype. Co-segregation studies and confirmation of variants identified by NGS were performed where possible. Functional genomics was performed as required. RESULTS: Of the 190 probands, 81 received an accurate genetic diagnosis. All except two of these cases harboured class 4 and/or 5 variants based on the American College of Medical Genetics and Genomics guidelines. We identified phenotypic expansions associated with CACNA1S, CHRNB1, GMPPB and STAC3. We describe a total of 50 novel variants, including a novel missense variant in the recently identified gene for arthrogryposis with brain malformations-SMPD4. CONCLUSIONS: Comprehensive gene panels give a diagnosis for a substantial proportion (42%) of fetal akinesia and arthrogryposis cases, even in an unselected cohort. Recently identified genes account for a relatively large proportion, 32%, of the diagnoses. Diagnostic-research collaboration was critical to the diagnosis and variant interpretation in many cases, facilitated genotype-phenotype expansions and reclassified VUS through functional genomics.

Mutations in ATP1A1 Cause Dominant Charcot-Marie-Tooth Type 2.

Although mutations in more than 90 genes are known to cause CMT, the underlying genetic cause of CMT remains unknown in more than 50% of affected individuals. The discovery of additional genes that harbor CMT2-causing mutations increasingly depends on sharing sequence data on a global level. In this way-by combining data from seven countries on four continents-we were able to define mutations in ATP1A1, which encodes the alpha1 subunit of the Na+,K+-ATPase, as a cause of autosomal-dominant CMT2. Seven missense changes were identified that segregated within individual pedigrees: c.143T>G (p.Leu48Arg), c.1775T>C (p.Ile592Thr), c.1789G>A (p.Ala597Thr), c.1801_1802delinsTT (p.Asp601Phe), c.1798C>G (p.Pro600Ala), c.1798C>A (p.Pro600Thr), and c.2432A>C (p.Asp811Ala). Immunostaining peripheral nerve axons localized ATP1A1 to the axolemma of myelinated sensory and motor axons and to Schmidt-Lanterman incisures of myelin sheaths. Two-electrode voltage clamp measurements on Xenopus oocytes demonstrated significant reduction in Na+ current activity in some, but not all, ouabain-insensitive ATP1A1 mutants, suggesting a loss-of-function defect of the Na+,K+ pump. Five mutants fall into a remarkably narrow motif within the helical linker region that couples the nucleotide-binding and phosphorylation domains. These findings identify a CMT pathway and a potential target for therapy development in degenerative diseases of peripheral nerve axons.

A novel RFC1 repeat motif (ACAGG) in two Asia-Pacific CANVAS families.

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) is a progressive late-onset, neurological disease. Recently, a pentanucleotide expansion in intron 2 of RFC1 was identified as the genetic cause of CANVAS. We screened an Asian-Pacific cohort for CANVAS and identified a novel RFC1 repeat expansion motif, (ACAGG)exp, in three affected individuals. This motif was associated with additional clinical features including fasciculations and elevated serum creatine kinase. These features have not previously been described in individuals with genetically-confirmed CANVAS. Haplotype analysis showed our patients shared the same core haplotype as previously published, supporting the possibility of a single origin of the RFC1 disease allele. We analysed data from >26 000 genetically diverse individuals in gnomAD to show enrichment of (ACAGG) in non-European populations.

Cerebellar ataxia, neuropathy, vestibular areflexia syndrome due to RFC1 repeat expansion.

Ataxia, causing imbalance, dizziness and falls, is a leading cause of neurological disability. We have recently identified a biallelic intronic AAGGG repeat expansion in replication factor complex subunit 1 (RFC1) as the cause of cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) and a major cause of late onset ataxia. Here we describe the full spectrum of the disease phenotype in our first 100 genetically confirmed carriers of biallelic repeat expansions in RFC1 and identify the sensory neuropathy as a common feature in all cases to date. All patients were Caucasian and half were sporadic. Patients typically reported progressive unsteadiness starting in the sixth decade. A dry spasmodic cough was also frequently associated and often preceded by decades the onset of walking difficulty. Sensory symptoms, oscillopsia, dysautonomia and dysarthria were also variably associated. The disease seems to follow a pattern of spatial progression from the early involvement of sensory neurons, to the later appearance of vestibular and cerebellar dysfunction. Half of the patients needed walking aids after 10 years of disease duration and a quarter were wheelchair dependent after 15 years. Overall, two-thirds of cases had full CANVAS. Sensory neuropathy was the only manifestation in 15 patients. Sixteen patients additionally showed cerebellar involvement, and six showed vestibular involvement. The disease is very likely to be underdiagnosed. Repeat expansion in RFC1 should be considered in all cases of sensory ataxic neuropathy, particularly, but not only, if cerebellar dysfunction, vestibular involvement and cough coexist.

Genomic sequencing highlights the diverse molecular causes of Perrault syndrome: a peroxisomal disorder (PEX6), metabolic disorders (CLPP, GGPS1), and mtDNA maintenance/translation disorders (LARS2, TFAM).

Perrault syndrome is a rare heterogeneous condition characterised by sensorineural hearing loss and premature ovarian insufficiency. Additional neuromuscular pathology is observed in some patients. There are six genes in which variants are known to cause Perrault syndrome; however, these explain only a minority of cases. We investigated the genetic cause of Perrault syndrome in seven affected individuals from five different families, successfully identifying the cause in four patients. This included previously reported and novel causative variants in known Perrault syndrome genes, CLPP and LARS2, involved in mitochondrial proteolysis and mitochondrial translation, respectively. For the first time, we show that pathogenic variants in PEX6 can present clinically as Perrault syndrome. PEX6 encodes a peroxisomal biogenesis factor, and we demonstrate evidence of peroxisomal dysfunction in patient serum. This study consolidates the clinical overlap between Perrault syndrome and peroxisomal disorders, and highlights the need to consider ovarian function in individuals with atypical/mild peroxisomal disorders. The remaining patients had variants in candidate genes such as TFAM, involved in mtDNA transcription, replication, and packaging, and GGPS1 involved in mevalonate/coenzyme Q10 biosynthesis and whose enzymatic product is required for mouse folliculogenesis. This genomic study highlights the diverse molecular landscape of this poorly understood syndrome.

New era in genetics of early-onset muscle disease: Breakthroughs and challenges.

Early-onset muscle disease includes three major entities that present generally at or before birth: congenital myopathies, congenital muscular dystrophies and congenital myasthenic syndromes. Almost exclusively there is weakness and hypotonia, although cases manifesting hypertonia are increasingly being recognised. These diseases display a wide phenotypic and genetic heterogeneity, with the uptake of next generation sequencing resulting in an unparalleled extension of the phenotype-genotype correlations and "diagnosis by sequencing" due to unbiased sequencing. Perhaps now more than ever, detailed clinical evaluations are necessary to guide the genetic diagnosis; with arrival at a molecular diagnosis frequently occurring following dialogue between the molecular geneticist, the referring clinician and the pathologist. There is an ever-increasing blurring of the boundaries between the congenital myopathies, dystrophies and myasthenic syndromes. In addition, many novel disease genes have been described and new insights have been gained into skeletal muscle development and function. Despite the advances made, a significant percentage of patients remain without a molecular diagnosis, suggesting that there are many more human disease genes and mechanisms to identify. It is now technically- and clinically-feasible to perform next generation sequencing for severe diseases on a population-wide scale, such that preconception-carrier screening can occur. Newborn screening for selected early-onset muscle diseases is also technically and ethically-achievable, with benefits to the patient and family from early management of these diseases and should also be implemented. The need for world-wide Reference Centres to meticulously curate polymorphisms and mutations within a particular gene is becoming increasingly apparent, particularly for interpretation of variants in the large genes which cause early-onset myopathies: NEB, RYR1 and TTN. Functional validation of candidate disease variants is crucial for accurate interpretation of next generation sequencing and appropriate genetic counseling. Many published "pathogenic" variants are too frequent in control populations and are thus likely rare polymorphisms. Mechanisms need to be put in place to systematically update the classification of variants such that accurate interpretation of variants occurs. In this review, we highlight the recent advances made and the challenges ahead for the molecular diagnosis of early-onset muscle diseases.

Congenital Titinopathy: Comprehensive characterization and pathogenic insights.

OBJECTIVE: Comprehensive clinical characterization of congenital titinopathy to facilitate diagnosis and management of this important emerging disorder. METHODS: Using massively parallel sequencing we identified 30 patients from 27 families with 2 pathogenic nonsense, frameshift and/or splice site TTN mutations in trans. We then undertook a detailed analysis of the clinical, histopathological and imaging features of these patients. RESULTS: All patients had prenatal or early onset hypotonia and/or congenital contractures. None had ophthalmoplegia. Scoliosis and respiratory insufficiency typically developed early and progressed rapidly, whereas limb weakness was often slowly progressive, and usually did not prevent independent walking. Cardiac involvement was present in 46% of patients. Relatives of 2 patients had dilated cardiomyopathy. Creatine kinase levels were normal to moderately elevated. Increased fiber size variation, internalized nuclei and cores were common histopathological abnormalities. Cap-like regions, whorled or ring fibers, and mitochondrial accumulations were also observed. Muscle magnetic resonance imaging showed gluteal, hamstring and calf muscle involvement. Western blot analysis showed a near-normal sized titin protein in all samples. The presence of 2 mutations predicted to impact both N2BA and N2B cardiac isoforms appeared to be associated with greatest risk of cardiac involvement. One-third of patients had 1 mutation predicted to impact exons present in fetal skeletal muscle, but not included within the mature skeletal muscle isoform transcript. This strongly suggests developmental isoforms are involved in the pathogenesis of this congenital/early onset disorder. INTERPRETATION: This detailed clinical reference dataset will greatly facilitate diagnostic confirmation and management of patients, and has provided important insights into disease pathogenesis. Ann Neurol 2018;83:1105-1124.

Expanding the phenotype of GMPPB mutations.

Dystroglycanopathies are a heterogeneous group of diseases with a broad phenotypic spectrum ranging from severe disorders with congenital muscle weakness, eye and brain structural abnormalities and intellectual delay to adult-onset limb-girdle muscular dystrophies without mental retardation. Most frequently the disease onset is congenital or during childhood. The exception is FKRP mutations, in which adult onset is a common presentation. Here we report eight patients from five non-consanguineous families where next generation sequencing identified mutations in the GMPPB gene. Six patients presented as an adult or adolescent-onset limb-girdle muscular dystrophy, one presented with isolated episodes of rhabdomyolysis, and one as a congenital muscular dystrophy. This report expands the phenotypic spectrum of GMPPB mutations to include limb-girdle muscular dystrophies with adult onset with or without intellectual disability, or isolated rhabdomyolysis.

Novel CHKB mutation expands the megaconial muscular dystrophy phenotype.

INTRODUCTION: Mutations in the choline kinase beta (CHKB) gene are associated with a congenital muscular dystrophy with giant mitochondria at the periphery of muscle fibers. METHODS: We describe a patient of Italian origin in whom whole-exome sequencing revealed a novel homozygous nonsense mutation, c.648C>A, p.(Tyr216*), in exon 5 of CHKB. RESULTS: The patient presented with limb-girdle weakness and hypotonia from birth with mental retardation, and had sudden and transient deteriorations of muscle strength with acute intercurrent illnesses. Previously undescribed sarcolemmal overexpression of utrophin was noted in the muscle biopsy. CONCLUSIONS: Pathological features broaden the description of the entity and provide new insight in the pathogenic mechanisms. This case highlights the usefulness of next-generation sequencing in the diagnosis of rare and incompletely understood conditions.

Novel mutations widen the phenotypic spectrum of slow skeletal/ß-cardiac myosin (MYH7) distal myopathy.

Laing early onset distal myopathy and myosin storage myopathy are caused by mutations of slow skeletal/ß-cardiac myosin heavy chain encoded by the gene MYH7, as is a common form of familial hypertrophic/dilated cardiomyopathy. The mechanisms by which different phenotypes are produced by mutations in MYH7, even in the same region of the gene, are not known. To explore the clinical spectrum and pathobiology, we screened the MYH7 gene in 88 patients from 21 previously unpublished families presenting with distal or generalized skeletal muscle weakness, with or without cardiac involvement. Twelve novel mutations have been identified in thirteen families. In one of these families, the father of the proband was found to be a mosaic for the MYH7 mutation. In eight cases, de novo mutation appeared to have occurred, which was proven in four. The presenting complaint was footdrop, sometimes leading to delayed walking or tripping, in members of 17 families (81%), with other presentations including cardiomyopathy in infancy, generalized floppiness, and scoliosis. Cardiac involvement as well as skeletal muscle weakness was identified in nine of 21 families. Spinal involvement such as scoliosis or rigidity was identified in 12 (57%). This report widens the clinical and pathological phenotypes, and the genetics of MYH7 mutations leading to skeletal muscle diseases.

Two Novel Variants in PI4KA in a Family Presenting With Hereditary Spastic Paraparesis: A Case Report.

Objectives: To report novel biallelic PI4KA variants in a family presenting with pure hereditary spastic paraparesis. Methods: Two affected sisters presented with unsolved hereditary spastic paraparesis and underwent clinical and imaging assessments. This was followed by short-read next-generation sequencing. Results: Analysis of next-generation sequencing data uncovered compound heterozygous variants in PI4KA (NM_058004.4: c.[3883C>A];[5785A>C]; p.[(His1295Asn);(Thr1929Pro)]. Using ACMG guidelines, both variants were classified as likely pathogenic. Discussion: Here, next-generation sequencing revealed 2 novel compound heterozygous variants in the phosphatidylinositol 4-kinase alpha gene (PI4KA) in 2 sisters presenting with progressive pure hereditary spastic paraparesis. Pathogenic variants in PI4KA have previously been associated with a spectrum of disorders including autosomal recessive perisylvian polymicrogyria, with cerebellar hypoplasia, arthrogryposis, and pure spastic paraplegia. The cases presented in this study expand the phenotypic spectrum associated with PI4KA variants and contribute new likely pathogenic variants for testing in patients with otherwise unsolved hereditary spastic paraparesis.

POLR3A-related disorders: From spastic ataxia to generalised dystonia and long-term efficacy of deep brain stimulation.

While biallelic POLR3A loss-of-function variants are traditionally linked to hypomyelinating leukodystrophy, patients with a specific splice variant c.1909+22G>A manifest as adolescent-onset spastic ataxia without overt leukodystrophy. In this study, we reported eight new cases, POLR3A-related disorder with c.1909+22 variant. One of these patients showed expanded phenotypic spectrum of generalised dystonia and her sister remained asymptomatic except for hypodontia. Two patients with dystonic arm tremor responded to deep brain stimulation. In our systemic literature review, we found that POLR3A-related disorder with c.1909+22 variant has attenuated disease severity but frequency of dystonia and upper limb tremor did not differ among genotypes.

Neuroradiological findings in GAA-FGF14 ataxia (SCA27B): more than cerebellar atrophy.

Background: GAA-FGF14 ataxia (SCA27B) is a recently reported late-onset ataxia caused by a GAA repeat expansion in intron 1 of the FGF14 gene. Initial studies revealed cerebellar atrophy in 74-97% of patients. A more detailed brain imaging characterization of GAA-FGF14 ataxia is now needed to provide supportive diagnostic features and earlier disease recognition. Methods: We performed a retrospective review of the brain MRIs of 35 patients (median age at MRI 63 years; range 28-88 years) from Quebec (n=27), Nancy (n=3), Perth (n=3) and Bengaluru (n=2) to assess the presence of atrophy in vermis, cerebellar hemispheres, brainstem, cerebral hemispheres, and corpus callosum, as well as white matter involvement. Following the identification of the superior cerebellar peduncles (SCPs) involvement, we verified its presence in 54 GAA-FGF14 ataxia patients from four independent cohorts (Tübingen n=29; Donostia n=12; Innsbruck n=7; Cantabria n=6). To assess lobular atrophy, we performed quantitative cerebellar segmentation in 5 affected subjects with available 3D T1-weighted images and matched controls. Results: Cerebellar atrophy was documented in 33 subjects (94.3%). We observed SCP involvement in 22 subjects (62.8%) and confirmed this finding in 30/54 (55.6%) subjects from the validation cohorts. Cerebellar segmentation showed reduced mean volumes of lobules X and IV in the 5 affected individuals. Conclusions: Cerebellar atrophy is a key feature of GAA-FGF14 ataxia. The frequent SCP involvement observed in different cohorts may facilitate the diagnosis. The predominant involvement of lobule X correlates with the frequently observed downbeat nystagmus.

A common flanking variant is associated with enhanced stability of the FGF14-SCA27B repeat locus.

The factors driving or preventing pathological expansion of tandem repeats remain largely unknown. Here, we assessed the FGF14 (GAA)·(TTC) repeat locus in 2,530 individuals by long-read and Sanger sequencing and identified a common 5'-flanking variant in 70.34% of alleles analyzed (3,463/4,923) that represents the phylogenetically ancestral allele and is present on all major haplotypes. This common sequence variation is present nearly exclusively on nonpathogenic alleles with fewer than 30 GAA-pure triplets and is associated with enhanced stability of the repeat locus upon intergenerational transmission and increased Fiber-seq chromatin accessibility.

Dominant mutations in KBTBD13, a member of the BTB/Kelch family, cause nemaline myopathy with cores.

We identified a member of the BTB/Kelch protein family that is mutated in nemaline myopathy type 6 (NEM6), an autosomal-dominant neuromuscular disorder characterized by the presence of nemaline rods and core lesions in the skeletal myofibers. Analysis of affected families allowed narrowing of the candidate region on chromosome 15q22.31, and mutation screening led to the identification of a previously uncharacterized gene, KBTBD13, coding for a hypothetical protein and containing missense mutations that perfectly cosegregate with nemaline myopathy in the studied families. KBTBD13 contains a BTB/POZ domain and five Kelch repeats and is expressed primarily in skeletal and cardiac muscle. The identified disease-associated mutations, C.742C>A (p.Arg248Ser), c.1170G>C (p.Lys390Asn), and c.1222C>T (p.Arg408Cys), located in conserved domains of Kelch repeats, are predicted to disrupt the molecule's beta-propeller blades. Previously identified BTB/POZ/Kelch-domain-containing proteins have been implicated in a broad variety of biological processes, including cytoskeleton modulation, regulation of gene transcription, ubiquitination, and myofibril assembly. The functional role of KBTBD13 in skeletal muscle and the pathogenesis of NEM6 are subjects for further studies.

Spinocerebellar ataxia 27B: episodic symptoms and acetazolamide response in 34 patients.

Ashton C et al report a retrospective multi-centre cohort of 34 patients from Canada, France, Austria and Australia with spinocerebellar ataxia 27B, describing the common feature of episodic ataxia and other episodic features, as well as the inefficacy of acetazolamide in these patients.

Uncovering the significance of expanded CD8+ large granular lymphocytes in inclusion body myositis: Insights into T cell phenotype and functional alterations, and disease severity.

Introduction: Inclusion body myositis (IBM) is a progressive inflammatory myopathy characterised by skeletal muscle infiltration and myofibre invasion by CD8+ T lymphocytes. In some cases, IBM has been reported to be associated with a systemic lymphoproliferative disorder of CD8+ T cells exhibiting a highly differentiated effector phenotype known as T cell Large Granular Lymphocytic Leukemia (T-LGLL). Methods: We investigated the incidence of a CD8+ T-LGL lymphoproliferative disorder in 85 IBM patients and an aged-matched group of 56 Healthy Controls (HC). Further, we analysed the phenotypical characteristics of the expanded T-LGLs and investigated whether their occurrence was associated with any particular HLA alleles or clinical characteristics. Results: Blood cell analysis by flow cytometry revealed expansion of T-LGLs in 34 of the 85 (40%) IBM patients. The T cell immunophenotype of T-LGLHIGH patients was characterised by increased expression of surface molecules including CD57 and KLRG1, and to a lesser extent of CD94 and CD56 predominantly in CD8+ T cells, although we also observed modest changes in CD4+ T cells and γδ T cells. Analysis of Ki67 in CD57+ KLRG1+ T cells revealed that only a small proportion of these cells was proliferating. Comparative analysis of CD8+ and CD4+ T cells isolated from matched blood and muscle samples donated by three patients indicated a consistent pattern of more pronounced alterations in muscles, although not significant due to small sample size. In the T-LGLHIGH patient group, we found increased frequencies of perforin-producing CD8+ and CD4+ T cells that were moderately correlated to combined CD57 and KLRG1 expression. Investigation of the HLA haplotypes of 75 IBM patients identified that carriage of the HLA-C*14:02:01 allele was significantly higher in T-LGLHIGH compared to T-LGLLOW individuals. Expansion of T-LGL was not significantly associated with seropositivity patient status for anti-cytosolic 5'-nucleotidase 1A autoantibodies. Clinically, the age at disease onset and disease duration were similar in the T-LGLHIGH and T-LGLLOW patient groups. However, metadata analysis of functional alterations indicated that patients with expanded T-LGL more frequently relied on mobility aids than T-LGLLOW patients indicating greater disease severity. Conclusion: Altogether, these results suggest that T-LGL expansion occurring in IBM patients is correlated with exacerbated immune dysregulation and increased disease burden.