TRPV4 mutations and cytotoxic hypercalcemia in axonal Charcot-Marie-Tooth neuropathies.

OBJECTIVE: To improve understanding of TRPV4-associated axonal Charcot-Marie-Tooth (CMT) neuropathy phenotypes and their debated pathologic mechanism. METHODS: A total of 17 CMT2C phenotypic families with vocal cord and diaphragmatic involvement and 36 clinically undifferentiated CMT2 subjects underwent sequencing analysis of the coding region of TRPV4. Functional studies of mutant proteins were performed using transiently transfected cells for TRPV4 subcellular localization, basal and stimulated Ca(2+) channel analysis, and cell viability assay with or without channel blockade. RESULTS: Two TRPV4 mutations R232C and R316H from 17 CMT2C families were identified in the ankyrin repeat domains. The R316H is a novel de novo mutation found in a patient with CMT2C phenotype. The family with R232C mutation had individuals with and without vocal cord and diaphragm involvement. Both mutant TRPV4 proteins had normal subcellular localization in HEK293 and HeLa cells. Cells transfected with R232C and R316H displayed increased intracellular Ca(2+) levels and reversible cell death by the TRPV channel antagonist, ruthenium red. CONCLUSION: TRPV4 ankyrin domain alterations including a novel de novo mutation cause axonal CMT2. Individuals with the same mutation may have nondistinct CMT2 or have phenotypic CMT2C with vocal cord paresis. Reversible hypercalcemic gain-of-function of mutant TRPV4 instead of loss-of-function appears to be pathologically important. The reversibility of cell death by channel blockade provides an attractive area of investigation in consideration of treatable axonal degeneration.

Mutations in FAM20C also identified in non-lethal osteosclerotic bone dysplasia.

Raine syndrome is an osteosclerotic bone dysplasia, which has proved to be lethal within the first few weeks of life in all the reported cases to date. We recently identified a chromosomal rearrangement and telomeric microdeletion in a patient with Raine syndrome and subsequently identified mutations in the FAM20C gene, located within the deleted region, in six additional Raine syndrome cases. The phenotype of Raine syndrome in the cases examined was remarkably consistent with generalized osteosclerosis of all bones, periosteal bone formation, characteristic facial phenotype and lethal within the first few weeks of life. In the current study, we have identified two unrelated individuals who presented at birth with a sclerosing bone dysplasia with features very similar to those in Raine syndrome but who survived infancy and are now aged 8 and 11 years, respectively. Mutations in FAM20C, consistent with autosomal recessive inheritance, were identified in both cases. In the first case, a homozygous non-synonymous mutation in exon 7 (1309G>A D437N) was identified, and in the second case, compound heterozygosity for non-synonymous mutations in exon 2 (731T>A I244N) and in exon 3 (796G>A G266R) was revealed. Raine syndrome has been previously considered to be a neonatal lethal condition. However, the identification of mutations in these two patients confirms a broader phenotypic spectrum and that mutation of FAM20C does not always lead to the infantile lethality previously seen as a prerequisite for Raine syndrome diagnosis.

Homozygous mutation of desmocollin-2 in arrhythmogenic right ventricular cardiomyopathy with mild palmoplantar keratoderma and woolly hair.

OBJECTIVES: The phenotypic triad of arrhythmogenic right ventricular cardiomyopathy (ARVC) associated with palmoplantar keratoderma and woolly hair has been previously associated with homozygous mutations in both plakoglobin and desmoplakin, which are both critical components of the desmosome. We present here a clinical and genetic study of a consanguineous pedigree in which 2 siblings present with ARVC with left ventricular involvement and associated mild palmoplantar keratoderma and woolly hair. METHODS: Clinical evaluation of the 2 patients and their family members was undertaken along with a homozygosity-mapping approach to identify the relevant gene and sequencing analysis to identify the causative mutation. RESULTS: The homozygosity-mapping approach excluded the involvement of both plakoglobin and desmoplakin in this pedigree. However, an extended region of homozygosity in both affected cases was revealed at the chromosome 18 desmocollin/desmoglein cluster, genes which encode components of the desmosome. Sequence analysis of the democollin-2 gene, located within this cluster, revealed a homozygous single-base deletion in exon 12 (1841delG). This mutation is predicted to lead to a frame shift and a premature termination codon at position 625 (S614fsX625). CONCLUSIONS: This is the first reported case of a mutation in desmocollin-2 associated with autosomal recessive ARVC.

A mutation in NFkappaB interacting protein 1 causes cardiomyopathy and woolly haircoat syndrome of Poll Hereford cattle.

Cardiomyopathy and woolly haircoat syndrome (CWH) of Poll Hereford cattle is a lethal, autosomal recessive disorder. Cardiac and haircoat changes are congenital, neonatal ocular keratitis develops in some cases and death usually occurs within the first 12 weeks of life. We undertook a homozygosity mapping approach to identify the chromosomal location of the causative gene. Seven candidate genes were examined for homozygosity in affected animals: desmoplakin and junction plakoglobin (both previously implicated in human cardiocutaneous syndromes), desmocollin 2, desmoglein 2, plakophilin 2, nuclear factor kappa B (NFKB1) and NFkappaB interacting protein 1 (PPP1R13L, also known as NKIP1). Homozygosity in 13 affected animals was observed at the PPP1R13L locus, located on bovine chromosome 18. Subsequent sequence analysis revealed a 7-bp duplication (c.956_962dup7) in exon 6 of this 13-exon gene. This frameshift variant is predicted to result in the substitution of three amino acids and the introduction of a premature stop codon at position 325 of the protein product (p.Ser322GlnfsX4). PPP1R13L interacts with NFkappaB, a family of structurally related transcription factors that regulate genes controlling inflammation, immune responses and cell proliferation and survival. CWH represents a large-animal model for cardiocutaneous disorders caused by a mutation in the PPP1R13L gene. The identification of this bovine mutation also indicates that PPP1R13L and other genes affecting NFkappaB activity may be candidate genes in the study of human cardiovascular disease.

Homozygous mutation of MYBPC3 associated with severe infantile hypertrophic cardiomyopathy at high frequency among the Amish.

BACKGROUND: Familial hypertrophic cardiomyopathy (HCM) is a leading cause of sudden cardiac death among young and apparently healthy people. Autosomal dominant mutations within genes encoding sarcomeric proteins have been identified. An autosomal recessive form of HCM has been discovered in a group of Amish children that is associated with poor prognosis and death within the first year of life. Affected patients experienced progressive cardiac failure despite maximal medical treatment. Postmortem histology showed myofibre disarray and myocyte loss consistent with refractory clinical deterioration in affected infants. OBJECTIVE: To conduct a genome-wide screen for linkage and try to identify an autozygous region which cosegregates with the infant cardiac phenotype METHODS AND RESULTS: An autozygous region of chromosome 11 which cosegregates with the infant cardiac phenotype was identified. This region contained the MYBPC3 gene, which has previously been associated with autosomal dominant adult-onset HCM. Sequence analysis of the MYBPC3 gene identified a splice site mutation in intron 30 which was homozygous in all affected infants. All surviving patients with the homozygous MYBPC3 gene mutations (3330+2T>G) underwent an orthotopic heart transplantation. CONCLUSIONS: Homozygous mutations in the MYBPC3 gene have been identified as the cause of severe infantile HCM among the Amish population.

A novel locus for an autosomal recessive hereditary spastic paraplegia (SPG35) maps to 16q21-q23.

BACKGROUND: The hereditary spastic paraplegias (HSPs) are a group of clinically and genetically heterogeneous neurodegenerative disorders in which the cardinal pathologic feature is upper motor neuron degeneration leading to progressive spasticity and weakness of the lower limbs. To date, 14 autosomal recessive HSP loci have been mapped. METHODS: We have identified a large consanguineous Omani family in which an autosomal recessive form of HSP is segregating. The age at onset varied from 6 to 11 years and the course of the disease is progressive with intellectual disability and is associated with seizures in two individuals. To map the chromosomal location of the causative gene we undertook 250K gene chip SNP analyses of all affected individuals assuming that a founder mutation was responsible. RESULTS: All affected individuals shared a 20.4 Mb (3.25 cM) region of homozygosity located on chromosome 16q21-q23.1, defined by SNP markers rs149428 and rs9929635 (peak multipoint lod score of 4.86). Two candidate genes, dynein, cytoplasmic 1, light intermediate chain 2 (DYNC1LI2) and vacuolar protein sorting 4 homolog A (VPS4A), were sequenced but no disease causing mutations were identified. CONCLUSION: We have mapped the chromosomal location of a novel gene responsible for a form of hereditary spastic paraplegia (HSP) (SPG35) and defined its clinical presentation.

Mutations in FAM20C are associated with lethal osteosclerotic bone dysplasia (Raine syndrome), highlighting a crucial molecule in bone development.

The generation and homeostasis of bone tissue throughout development and maturity is controlled by the carefully balanced processes of bone formation and resorption. Disruption of this balance can give rise to a broad range of skeletal pathologies. Lethal osteosclerotic bone dysplasia (or, Raine syndrome) is an autosomal recessive disorder characterized by generalized osteosclerosis with periosteal bone formation and a distinctive facial phenotype. Affected individuals survive only days or weeks. We have identified and defined a chromosome 7 uniparental isodisomy and a 7p telomeric microdeletion in an affected subject. The extent of the deleted region at the 7p telomere was established by genotyping microsatellite markers across the telomeric region. The region is delimited by marker D7S2563 and contains five transcriptional units. Sequence analysis of FAM20C, located within the deleted region, in six additional affected subjects revealed four homozygous mutations and two compound heterozygotes. The identified mutations include four nonsynonymous base changes, all affecting evolutionarily conserved residues, and four splice-site changes that are predicted to have a detrimental effect on splicing. FAM20C is a member of the FAM20 family of secreted proteins, and its mouse orthologue (DMP4) has demonstrated calcium-binding properties; we also show by in situ hybridization its expression profile in mineralizing tissues during development. This study defines the causative role of FAM20C in this lethal osteosclerotic disorder and its crucial role in normal bone development.

The natural history of Noonan syndrome: a long-term follow-up study.

OBJECTIVE: To define better the adult phenotype and natural history of Noonan syndrome. DESIGN: A prospective observational study of a large cohort. RESULTS: Data are presented for 112 individuals with Noonan syndrome (mean age 25.3 (range 12-71) years), who were followed up for a mean of 12.02 years. Mutations in PTPN11 were identified in 35% of probands. Ten subjects died during the study interval; three of these deaths were secondary to heart failure associated with hypertrophic cardiomyopathy. Pulmonary stenosis affected 73 (65%) subjects; 42 (58%) required no intervention, nine underwent balloon pulmonary valvuloplasty (three requiring further intervention) and 22 surgical valvuloplasty (three requiring further intervention). Hypertrophic cardiomyopathy affected 21 (19%) patients, which had remitted in two cases, but one subject required cardiac transplant. No subjects died suddenly or had symptoms suggestive of arrhythmia. The mean final adult height was 167.4 cm in males and 152.7 cm in females. Feeding problems in infancy were identified as a predictor of future outcome. The mean age of speaking in two-word phrases was 26 months for those with no feeding difficulties, compared with 39 months for those with severe problems requiring nasogastric feeding. Attendance at a school for children with special needs for the same groups was 12.5% and 58%, respectively. A statement of special educational need had been issued in 44% overall; however, academic achievement was broadly similar to that of the general population. IMPLICATIONS: Although the morbidity for some patients with Noonan syndrome is low, early predictors of poorer outcome have been identified, which will help ascertain those most in need of intervention.

Complicated hereditary spastic paraplegia with thin corpus callosum (HSP-TCC) and childhood onset.

The hereditary spastic paraplegias (HSPs) are a group of rare disorders with the predominant clinical feature of progressive spastic paraplegia. They are subdivided into pure and complicated forms according to whether the disorder is associated with other neurological abnormalities. We report on two unrelated female Caucasian patients with complicated HSP, aged 16 and 24 years, who showed progressive gait disturbance with spasticity and ataxia as well as cognitive impairment. Onset of symptoms was at age 3 and 10 years, respectively. MRI revealed mild diffuse non-progressive T (2)-signal alterations of cerebral white matter and thinning of the body and genu of the corpus callosum. Some similarity of clinical symptoms and MRI patterns with the phenotype of Mast syndrome prompted a mutation analysis of the SPG21 gene, encoding maspardin, which revealed a wild-type sequence in both patients. Clinical and neuroradiological features in our patients are diagnostic for complicated autosomal recessive hereditary spastic paraplegia with thin corpus callosum (HSP-TCC, SPG11). This disorder, characterized by a typical MRI pattern and a progressive spastic paraplegia that may be associated with dementia and ataxia, may have an onset in early childhood and probably is one of the more common forms of complicated HSP.

SPG3A mutation screening in English families with early onset autosomal dominant hereditary spastic paraplegia.

Mutations in the SPG3A gene encoding the novel GTPase atlastin have recently been implicated in causing autosomal dominant hereditary spastic paraplegia (ADHSP) in six unrelated families. The phenotype of affected individuals in all cases has been of an early onset uncomplicated form of the disease. One particular missense mutation, R239C, in exon 7 of SPG3A has been identified in three of these families. We performed mutation screening by direct sequencing of all 14 exons and flanking sequences of the SPG3A gene in affected individuals from 12 unrelated English families, all with an early onset uncomplicated ADHSP in whom spastin mutations had previously been excluded. The R239C mutation was found to co-segregate with the disease in one English ADHSP family confirming a widespread prevalence for this commonly occurring mutation. No additional SPG3A mutations were identified in the remaining 11 families suggesting that even within this specific sub-set of early onset uncomplicated ADHSP patients atlastin mutations are relatively rare.

A clinical and genetic study of SPG5A linked autosomal recessive hereditary spastic paraplegia.

The authors performed a clinical and genetic study of a large consanguineous English family with uncomplicated autosomal recessive hereditary spastic paraplegia (ARHSP). Linkage to the previously described SPG5A locus was established with maximum multipoint lod score of 4.84. The locus was refined to a 23.6 cM interval between markers D8S1833 and D8S285. No evidence of oxidative phosphorylation defects was found in muscle biopsies from two affected individuals.

Screening of patients with hereditary spastic paraplegia reveals seven novel mutations in the SPG4 (Spastin) gene.

Hereditary spastic paraplegia (HSP) is a heterogeneous condition characterised in its pure form by progressive lower limb spasticity. Mutations in SPG4 (encoding spastin) may be responsible for up to 40% of autosomal dominant (AD) cases. A cohort of 41 mostly pure HSP patients from Britain and Austria, 30 of whom displayed AD inheritance, was screened for mutations in SPG4 by single strand conformation polymorphism (SSCP) analysis followed by sequencing of samples with mobility shifts. We identified eight SPG4 mutations in pure AD HSP patients, seven of which were novel: one missense mutation within the AAA cassette (1633G>T), two splice site mutations (1130-1G>T, 1853+2T>A) and four frameshift mutations (190_208dup19, 1259_1260delGT, 1702_1705delGAAG, 1845delG). A novel duplication in intron 11 (1538+42_45dupTATA) was also detected. We report the HUGO-approved nomenclature of these mutations as well. Furthermore, we detected a silent change (1004G>A; P293P), previously reported as a mutation, which was also present in controls. The frequency of SPG4 mutations detected in pure AD HSP was 33.3%, suggesting that screening of such patients for SPG4 mutations is worthwhile. Most patients will have unique mutations. Screening of SPG4 in apparently isolated cases of HSP may be of less value.

Diagnostic strategies in CADASIL.

BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited autosomal dominant condition characterized by migraine, recurrent stroke, and dementia. It results from mutations in the notch3 gene but mutations may occur at multiple sites making molecular diagnosis time consuming. It has been suggested that the presence of granular osmiophilic material (GOM) on skin biopsy and involvement of the anterior temporal lobe and external capsule on MRI may help in diagnosis. METHODS: The authors identified 83 potential index cases from the British population and screened exons 2 to 23 of notch3. MRI scans were scored using a modified Scheltens scale. Skin biopsy was performed in a subgroup. RESULTS: Fifteen different point mutations were identified in 48 families, 73% of which were in exon 4, 8% in exon 3, and 6% in each of exons 5 and 6. Moderate or severe involvement of the anterior temporal pole on MRI had a sensitivity of 89% and specificity of 86% for diagnosis of CADASIL, whereas external capsule involvement had a high sensitivity of 93% but a low specificity of 45%. Skin biopsy, performed in 18 cases, had a sensitivity of 45% and specificity of 100%. CONCLUSIONS: The spectrum of mutations in this study can be used to plan appropriate screening protocols; a suggested protocol is to screen exon 4, and proceed to exons 3, 5, and 6 where indicated. GOM on skin biopsy is diagnostic but can be negative. Anterior temporal pole involvement on MRI is a useful diagnostic marker.

A large family with hereditary spastic paraparesis due to a frame shift mutation of the spastin (SPG4) gene: association with multiple sclerosis in two affected siblings and epilepsy in other affected family members.

Hereditary spastic paraparesis (HSP) is a clinically and genetically heterogeneous neurodegenerative disorder characterised by progressive lower limb spasticity and weakness. Some forms have been associated with white matter lesions and complex phenotypes. This study was prompted by the diagnosis of multiple sclerosis (MS) in two sisters from a large pedigree with hereditary spastic paraparesis. Twelve affected members of the extended family were examined (MRI and EEG were carried out and evoked potentials measured in five), and historical information was obtained from six affected deceased persons. The inherited disease phenotype was confirmed as autosomal dominant hereditary spastic paraparesis associated with epilepsy in four affected persons. None of the extended family had evidence of MS. Genetic analysis of the family has shown linkage to chromosome 2p and sequencing of the spastin gene has identified a 1406delT frameshift mutation in exon 10. This kindred demonstrates the clinical heterogeneity of HSP associated with spastin mutations. The possible relevance of the concurrence of HSP and MS in the sib pair is discussed.

Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome.

Noonan syndrome (MIM 163950) is an autosomal dominant disorder characterized by dysmorphic facial features, proportionate short stature and heart disease (most commonly pulmonic stenosis and hypertrophic cardiomyopathy). Webbed neck, chest deformity, cryptorchidism, mental retardation and bleeding diatheses also are frequently associated with this disease. This syndrome is relatively common, with an estimated incidence of 1 in 1,000-2,500 live births. It has been mapped to a 5-cM region (NS1) [corrected] on chromosome 12q24.1, and genetic heterogeneity has also been documented. Here we show that missense mutations in PTPN11 (MIM 176876)-a gene encoding the nonreceptor protein tyrosine phosphatase SHP-2, which contains two Src homology 2 (SH2) domains-cause Noonan syndrome and account for more than 50% of the cases that we examined. All PTPN11 missense mutations cluster in interacting portions of the amino N-SH2 domain and the phosphotyrosine phosphatase domains, which are involved in switching the protein between its inactive and active conformations. An energetics-based structural analysis of two N-SH2 mutants indicates that in these mutants there may be a significant shift of the equilibrium favoring the active conformation. This implies that they are gain-of-function changes and that the pathogenesis of Noonan syndrome arises from excessive SHP-2 activity.

Silver syndrome is not linked to any of the previously established autosomal dominant hereditary spastic paraplegia loci.

The hereditary spastic paraplegias are a clinically variable and genetically heterogeneous group of disorders characterized by progressive and lower limb spasticity and weakness. Silver syndrome (SS) is a particularly disabling autosomal dominant form of the disease in which there is associated wasting of the hand muscles. In view of the fact that genes for hereditary spastic paraplegia can produce highly variable phenotypes, the eight known autosomal dominant loci were investigated for linkage to Silver syndrome. Genotyping of these loci in two large multigenerational families was incompatible with linkage to any of these regions, suggesting that an additional locus is responsible for this syndrome.

The Silver syndrome variant of hereditary spastic paraplegia maps to chromosome 11q12-q14, with evidence for genetic heterogeneity within this subtype.

The hereditary spastic paraplegias (HSPs) are a complex group of neurodegenerative disorders characterized by lower-limb spasticity and weakness. Silver syndrome (SS) is a particularly disabling dominantly inherited form of HSP, complicated by amyotrophy of the hand muscles. Having excluded the multiple known HSP loci, we undertook a genomewide screen for linkage of SS in one large multigenerational family, which revealed evidence for linkage of the SS locus, which we have designated "SPG17," to chromosome 11q12-q14. Haplotype construction and analysis of recombination events permitted the minimal interval defining SPG17 to be refined to approximately 13 cM, flanked by markers D11S1765 and D11S4136. SS in a second family was not linked to SPG17, demonstrating further genetic heterogeneity in HSP, even within this clinically distinct subtype.

Homozygosity at chromosome 8qter in individuals affected by mal de Meleda (Meleda disease) originating from the island of Meleda.

BACKGROUND: The inherited palmoplantar keratodermas (PPKs) are a clinically heterogeneous group of disorders characterized by thickening of the skin of the palms and the soles. These diseases also exhibit genetic heterogeneity and many autosomal dominant and recessive forms have been described. Mal de Meleda (Meleda disease, MD) is an autosomal recessive form of PPK first described on the Dalmatian island of Meleda. A gene for MD has recently been assigned to the most telomeric portion of chromosome 8q using two large Algerian families. OBJECTIVES: To determine whether the same gene underlies the skin disease in Meleda islanders. METHODS: We have examined five affected individuals originating from the Dalmatian island itself for 8qter homozygosity. RESULTS: This region was found to be homozygous in all five affected individuals but in none of the 20 other unaffected family members examined. CONCLUSIONS: The current study confirms the localization of a gene for MD to 8qter using samples from the island of Meleda, highlighting the clinical and genetic homogeneity of this condition.