Targeted Next Generation Sequencing reveals previously unidentified TSC1 and TSC2 mutations.

BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in TSC1 and TSC2. Conventional DNA diagnostic screens identify a TSC1 or TSC2 mutation in 75 - 90% of individuals categorised with definite TSC. The remaining individuals either have a mutation that is undetectable using conventional methods, or possibly a mutation in another as yet unidentified gene. METHODS: Here we apply a targeted Next Generation Sequencing (NGS) approach to screen the complete TSC1 and TSC2 genomic loci in 7 individuals fulfilling the clinical diagnostic criteria for definite TSC in whom no TSC1 or TSC2 mutations were identified using conventional screening methods. RESULTS: We identified and confirmed pathogenic mutations in 3 individuals. In the remaining individuals we identified variants of uncertain clinical significance. The identified variants included mosaic changes, changes located deep in intronic sequences and changes affecting promoter regions that would not have been identified using exon-only based analyses. CONCLUSIONS: Targeted NGS of the TSC1 and TSC2 loci is a suitable method to increase the yield of mutations identified in the TSC patient population.

Autosomal recessive spinocerebellar ataxia 7 (SCAR7) is caused by variants in TPP1, the gene involved in classic late-infantile neuronal ceroid lipofuscinosis 2 disease (CLN2 disease).

Spinocerebellar ataxias are phenotypically, neuropathologically, and genetically heterogeneous. The locus of autosomal recessive spinocerebellar ataxia type 7 (SCAR7) was previously linked to chromosome band 11p15. We have identified TPP1 as the causative gene for SCAR7 by exome sequencing. A missense and a splice site variant in TPP1, cosegregating with the disease, were found in a previously described SCAR7 family and also in another patient with a SCAR7 phenotype. TPP1, encoding the tripeptidyl-peptidase 1 enzyme, is known as the causative gene for late infantile neuronal ceroid lipofuscinosis disease 2 (CLN2 disease). CLN2 disease is characterized by epilepsy, loss of vision, ataxia, and a rapidly progressive course, leading to early death. SCAR7 patients showed ataxia and low activity of tripeptidyl-peptidase 1, but no ophthalmologic abnormalities or epilepsy. Also, the slowly progressive evolution of the disease until old age and absence of ultra structural curvilinear profiles is different from the known CLN2 phenotypes. Our findings now expand the phenotypes related to TPP1-variants to SCAR7. In spite of the limited sample size and measurements, a putative genotype-phenotype correlation may be drawn: we hypothesize that loss of function variants abolishing TPP1 enzyme activity lead to CLN2 disease, whereas variants that diminish TPP1 enzyme activity lead to SCAR7.

Hypomorphic NOTCH3 alleles do not cause CADASIL in humans.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by stereotyped missense mutations in NOTCH3. Whether these mutations lead to the CADASIL phenotype via a neomorphic effect, or rather by a hypomorphic effect, is subject of debate. Here, we report two novel NOTCH3 mutations, both leading to a premature stop codon with predicted loss of NOTCH3 function. The first mutation, c.307C>T, p.Arg103*, was detected in two brothers aged 50 and 55 years, with a brain MRI and skin biopsy incompatible with CADASIL. The other mutation was found in a 40-year-old CADASIL patient compound heterozygous for a pathogenic NOTCH3 mutation (c.2129A>G, p.Tyr710Cys) and an intragenic frameshift deletion. The deletion was inherited from his father, who did not have the skin biopsy abnormalities seen in CADASIL patients. These individuals with rare NOTCH3 mutations indicate that hypomorphic NOTCH3 alleles do not cause CADASIL.

Autosomal dominant restless legs syndrome maps to chromosome 20p13 (RLS-5) in a Dutch kindred.

Six chromosomal loci have been mapped for restless legs syndrome (RLS) through family-based linkage analysis (RLS-1 to RLS-6), but confirmation has met with limited success, and causative mutations have not yet been identified. We ascertained a large multigenerational Dutch family with RLS of early onset (average 18 years-old). The clinical study included a follow-up of 2 years. To map the underlying genetic defect, we performed a genome-wide scan for linkage using high-density SNP microarrays. A single, strong linkage peak was detected on chromosome 20p13, under an autosomal-dominant model, in the region of the RLS-5 locus (maximum multipoint LOD score 3.02). Haplotype analysis refined the RLS-5 critical region from 5.2 to 4.5 megabases. In conclusion, we provide the first confirmation of the RLS-5 locus, and we reduce its critical region. The identification of the underlying mutation might reveal an important susceptibility gene for this common movement disorder.

Ectopia lentis et pupillae in four generations caused by novel mutations in the ADAMTSL4 gene.

OBJECTIVES: To identify the phenotype, genetic defect and inheritance pattern of ectopia lentis et pupillae (ELP) in a large Dutch family, previously diagnosed as presumed autosomal dominant ELP because of the occurrence of ELP in three generations. DESIGN: A clinical and genetic study of children and adults. PARTICIPANTS: Eight patients of the ELP family, including five new patients from the youngest generation. METHODS: Standard ophthalmological examinations were performed. For molecular genetic analysis, the coding region of ADAMTSL4 was sequenced. Main outcome measures were the ocular phenotype of the new ELP patients, the inheritance pattern and the identification of mutations in the ADAMTSL4 gene in the family. RESULTS: Of the eight patients with ectopia lentis, seven fulfilled the clinical diagnostic criteria of ELP. Molecular genetic analysis of these seven patients disclosed two novel mutations in the ADAMTSL4 gene: homozygous (p.Q752X/p.Q752X) in six patients and compound heterozygous (p.Q752X/p.Q758fs) in one patient. Heterozygosity in phenotypically normal parents proved autosomal recessive (AR) inheritance. The pseudodominant inheritance pattern can be explained by high carrier frequency in this small community and/or consanguinity. CONCLUSIONS: Patients from a family with ELP in four generations have AR ELP caused by novel mutations in ADAMTSL4. The clinical presentation of ELP can be variable, but all patients of our study with homozygous p.Q752X mutation have ectopia lentis and pupillary dysfunction in common.

Cerebral cavernous malformations: from molecular pathogenesis to genetic counselling and clinical management.

Cerebral cavernous (or capillary-venous) malformations (CCM) have a prevalence of about 0.1-0.5% in the general population. Genes mutated in CCM encode proteins that modulate junction formation between vascular endothelial cells. Mutations lead to the development of abnormal vascular structures.In this article, we review the clinical features, molecular and genetic basis of the disease, and management.

Characterisation of TSC1 promoter deletions in tuberous sclerosis complex patients.

Tuberous sclerosis complex (TSC), an autosomal dominant disorder, is a multisystem disease with manifestations in the central nervous system, kidneys, skin and/or heart. Most TSC patients carry a pathogenic mutation in either TSC1 or TSC2. All types of mutations, including large rearrangements, nonsense, missense and frameshift mutations, have been identified in both genes, although large rearrangements in TSC1 are scarce. In this study, we describe the identification and characterisation of eight large rearrangements in TSC1 using multiplex ligation-dependent probe amplification (MLPA) in a cohort of 327 patients, in whom no pathogenic mutation was identified after sequence analysis of both TSC1 and TSC2 and MLPA analysis of TSC2. In four families, deletions only affecting the non-coding exon 1 were identified. In one case, loss of TSC1 mRNA expression from the affected allele indicated that exon 1 deletions are inactivating mutations. Although the number of TSC patients with large rearrangements of TSC1 is small, these patients tend to have a somewhat milder phenotype compared with the group of patients with small TSC1 mutations.