Genotype-guided diagnostic reassessment after exome sequencing in neuromuscular disorders: experiences with a two-step approach.

BACKGROUND AND PURPOSE: Next-generation sequencing has greatly improved the diagnostic success rates for genetic neuromuscular disorders (NMDs). Nevertheless, most patients still remain undiagnosed, and there is a need to maximize the diagnostic yield. METHODS: A retrospective study was conducted on 72 patients with NMDs who underwent exome sequencing (ES), partly followed by genotype-guided diagnostic reassessment and secondary investigations. The diagnostic yields that would have been achieved by appropriately chosen narrow and comprehensive gene panels were also analysed. RESULTS: The initial diagnostic yield of ES was 30.6% (n = 22/72 patients). In an additional 15.3% of patients (n = 11/72) ES results were of unknown clinical significance. After genotype-guided diagnostic reassessment and complementary investigations, the yield was increased to 37.5% (n = 27/72). Compared to ES, targeted gene panels (<25 kilobases) reached a diagnostic yield of 22.2% (n = 16/72), whereas comprehensive gene panels achieved 34.7% (n = 25/72). CONCLUSION: Exome sequencing allows the detection of pathogenic variants missed by (narrowly) targeted gene panel approaches. Diagnostic reassessment after genetic testing further enhances the diagnostic outcomes for NMDs.

The pathogenic LRRK2 R1441C mutation induces specific deficits modeling the prodromal phase of Parkinson's disease in the mouse.

The aim of the present study was to further explore the in vivo function of the Leucine-rich repeat kinase 2 (LRRK2)-gene, which is mutated in certain familial forms of Parkinson's disease (PD). We generated a mouse model harboring the disease-associated point mutation R1441C in the GTPase domain of the endogenous murine LRRK2 gene (LRRK2 R1441C line) and performed a comprehensive analysis of these animals throughout lifespan in comparison with an existing knockdown line of LRRK2 (LRRK2 knockdown line). Animals of both lines do not exhibit severe motor dysfunction or pathological signs of neurodegeneration neither at young nor old age. However, at old age the homozygous LRRK2 R1441C animals exhibit clear phenotypes related to the prodromal phase of PD such as impairments in fine motor tasks, gait, and olfaction. These phenotypes are only marginally observable in the LRRK2 knockdown animals, possibly due to activation of compensatory mechanisms as suggested by in vitro studies of synaptic transmission. Thus, at the organismal level the LRRK2 R1441C mutation does not emerge as a loss of function of the protein, but induces mutation specific deficits. Furthermore, judged by the phenotypes presented, the LRRK2-R1441C knock-in line is a valid preclinical model for the prodromal phase of PD.

Hereditary spastic paraplegia caused by compound heterozygous mutations outside the motor domain of the KIF1A gene.

BACKGROUND AND PURPOSE: Hereditary spastic paraplegia is a clinically and genetically heterogeneous group of rare, inherited disorders causing an upper motor neuron syndrome with (complex) or without (pure) additional neurological symptoms. Mutations in the KIF1A gene have already been associated with recessive and dominant forms of hereditary spastic paraplegia (SPG30) in a few cases. METHODS: All family members included in the study were examined neurologically. Whole-exome sequencing was used in affected individuals to identify the responsible candidate gene. Conventional Sanger sequencing was conducted to validate familial segregation. RESULTS: A family of Macedonian origin with two affected siblings, one with slowly progressive and the other one with a more complex and rapidly progressing hereditary spastic paraplegia is reported. In both affected individuals, two novel pathogenic mutations outside the motor domain of the KIF1A gene were found (NM_001244008.1:c.2909G>A, p.Arg970His and c.1214dup, p.Asn405Lysfs*40) that segregate with the disease within the family establishing the diagnosis of autosomal recessive SPG30. CONCLUSIONS: This report provides the first evidence that mutations outside the motor domain of the gene can cause (recessive) SPG30 and extends the genotype-phenotype association for KIF1A-related diseases.

The c.65-2A>G splice site mutation is associated with a mild phenotype in Danon disease due to the transcription of normal LAMP2 mRNA.

Danon disease (DD) is a rare X-linked multisystem disorder caused by mutations of the LAMP2 gene and characterized by intellectual disability, skeletal myopathy and cardiomyopathy. The survival time is severely reduced. Contrasting with the usual disease course, we report on a family with an exceptionally mild phenotype of DD despite having two potentially damaging LAMP2 mutations. Using RNA-Seq analysis, we showed that a c.65-2A>G splice site mutation results in the tissue-specific production of four different transcripts including the full-length mRNA in muscle tissue but not in leukocytes. We confirmed our results by immunohistochemistry and immunoblotting, showing the detection of LAMP2 protein only in muscle. The second mutation (c.586A>T, p.T196S) has been reported before to have an uncertain clinical significance. In our patients, however, neither of the two mutations seem to have a high enough functional impact to cause a severe phenotype. Overall, our study reveals that alternative splicing is a potential mechanism in DD with underlying splice site mutations of the LAMP2 gene in order to rescue the full-length mRNA. Moreover, our report of a mild phenotype complements the DD spectrum, which is of great importance for a rare disease suspected to be underdiagnosed.

Analysis of four prevalent filaggrin mutations (R501X, 2282del4, R2447X and S3247X) in Austrian and German patients with atopic dermatitis.

BACKGROUND: Recently, mutations in the filaggrin gene (FLG) have been shown to be a major predisposing factor for atopic dermatitis (AD). OBJECTIVE: In this study, we evaluated the influence of four prevalent mutations (R501X, 2282del4, R2447X and S3247X) in a large cohort of 462 Austrian and German AD patients and in 402 control individuals. RESULTS: We found a strong association of the FLG mutations with AD. Subgroup analysis revealed a significantly higher proportion of patients with an early age of disease onset and significantly higher median serum IgE levels among mutation carriers. Furthermore, we observed an overrepresentation of null alleles in AD patients with concomitant asthma compared with those without this co-morbidity. CONCLUSION: Our data confirm and extend the knowledge of the influence of FLG mutations in AD.

Clinical heterogeneity in 3 unrelated families linked to VCP p.Arg159His.

BACKGROUND: Families associated with missense mutations in the valosin-containing protein (VCP) present with a rare autosomal dominant multisystem disorder of frontotemporal lobar degeneration (FTLD), inclusion body myopathy (IBM), and Paget disease of bone (PDB), referred to as IBMPFD. METHODS: We used exon-based genomic DNA sequencing to test for VCP mutations in 123 unrelated Belgian patients with FTLD and their relatives, and the absence of such mutations in 157 control individuals. We analyzed haplotype sharing among mutation carriers by genotyping 8 microsatellite markers in the VCP locus. We obtained family history and clinical and pathologic data using established diagnostic instruments. RESULTS: Mutation analysis of VCP identified 2 Belgian patients with FTLD carrying the p.Arg159His mutation, which segregated in their families. In one family, patients presented with FTLD only, whereas in the other family, patients developed FTLD, PDB, or both without signs of IBM for any of the mutation carriers. We had previously identified p.Arg159His in an Austrian family with patients exhibiting both IBM and PDB. Haplotype sharing analysis indicated that the 3 p.Arg159His families are unrelated. Clinical follow-up of the Austrian family identified dementia symptoms in 1 patient. Autopsy data of 3 patients of the 2 Belgian families revealed FTLD pathology with numerous ubiquitin-immunoreactive, intranuclear inclusions and dystrophic neurites staining positive for TDP-43 protein. CONCLUSIONS: In 3 unrelated families with IBMPFD segregating VCP p.Arg159His, we observed a high degree of clinical heterogeneity and variable penetrance of the 3 cardinal clinical phenotypes: inclusion body myopathy, Paget disease of bone, and frontotemporal lobar degeneration. In contrast, the neuropathologic phenotype was consistent with FTLD-TDP type 4.

Analysis of the prodynorphin promoter polymorphism in atopic dermatitis and disease-related pruritus.

Pruritus is one of the key symptoms in atopic dermatitis (AD). The prodynorphin polypeptide is a precursor protein of pruritus-modulating opioid peptides. It is encoded by the prodynorphin gene (PDYN). To investigate a possible correlation of PDYN promoter polymorphisms with intensity of pruritus in patients with AD, we genotyped 211 Austrian patients with AD and 197 nonatopic controls. No significant association of the PDYN promoter polymorphism with AD in general was found when patients with AD were compared with controls. The analysis of possible associations with pruritus intensity also showed no relevant difference in the allelic distribution between patients with different pruritus-score values. These data argue against an important role of the PDYN promoter polymorphism in AD in general and in the development of disease-related pruritus, although owing to our small sample size, a weak effect cannot be excluded. Additional studies are needed to further evaluate the influence of PDYN polymorphism in pruritus.

Replication of restless legs syndrome loci in three European populations.

BACKGROUND: Restless legs syndrome (RLS) is associated with common variants in three intronic and intergenic regions in MEIS1, BTBD9, and MAP2K5/LBXCOR1 on chromosomes 2p, 6p and 15q. METHODS: Our study investigated these variants in 649 RLS patients and 1230 controls from the Czech Republic (290 cases and 450 controls), Austria (269 cases and 611 controls) and Finland (90 cases and 169 controls). Ten single nucleotide polymorphisms (SNPs) within the three genomic regions were selected according to the results of previous genome-wide scans. Samples were genotyped using Sequenom platforms. RESULTS: We replicated associations for all loci in the combined samples set (rs2300478 in MEIS1, p = 1.26 x 10(-5), odds ratio (OR) = 1.47, rs3923809 in BTBD9, p = 4.11 x 10(-5), OR = 1.58 and rs6494696 in MAP2K5/LBXCOR1, p = 0.04764, OR = 1.27). Analysing only familial cases against all controls, all three loci were significantly associated. Using sporadic cases only, we could confirm the association only with BTBD9. CONCLUSION: Our study shows that variants in these three loci confer consistent disease risks in patients of European descent. Among the known loci, BTBD9 seems to be the most consistent in its effect on RLS across populations and is also most independent of familial clustering.

A splice site variant in the sodium channel gene SCN1A confers risk of febrile seizures.

OBJECTIVE: Our aim was to investigate whether the risk of febrile seizures is influenced by a common functional polymorphism in the sodium channel gene SCN1A. This single nucleotide polymorphism (IVS5N+5 G>A, rs3812718) was shown to modify the proportion of two alternative transcripts of the channel. METHODS: We performed an exploratory case-control association analysis in 90 adult epilepsy patients with childhood febrile seizures vs 486 epilepsy patients without a history of febrile seizures and also vs 701 population controls. In the replication step, we investigated children with febrile seizures without concomitant epilepsy at the time of their inclusion. We compared the genotypes of 55 of those children against population controls and performed a within-family association analysis in an additional 88 child-parent trios with febrile seizures. RESULTS: We observed a significant association of the splice-site interrupting A-allele with febrile seizures (p value in the exploratory step: 0.000017; joint p value of the replication: 0.00069). Our data suggest that the A-allele of this variant confers a threefold genotype relative risk in homozygotes and accounts for a population attributable fraction of up to 50% for the etiology of febrile seizures. CONCLUSIONS: The A-allele of the SCN1A single nucleotide polymorphism IVS5N+5 G>A (rs3812718) represents a common and relevant risk factor for febrile seizures. A limitation of the present study is that patients of the exploratory and replication steps differed in aspects of their phenotype (febrile seizures with and without additional epilepsy).

A novel mutation in the MFSD8 gene in late infantile neuronal ceroid lipofuscinosis.

Neuronal ceroid lipofuscinoses (NCL) are lysosomal storage disorders and constitute the most common group of progressive neurodegenerative diseases in childhood. Most NCLs are inherited in a recessive manner and are clinically characterised by a variable age at onset, epileptic seizures, psychomotor decline, visual impairment and premature death. To date, eight causative genes have been identified to underlie various clinical forms of NCL. We performed a genome-wide linkage analysis followed by sequencing the recently described NCL gene MFSD8 in three affected and three unaffected members of a consanguineous Egyptian family with an autosomal recessively inherited progressive neurodegenerative disorder. The clinical picture of the patients was compatible with a late infantile NCL (LINCL); however, impairment of the visual system was not a cardinal symptom in the respective family. By linkage analysis, we identified two putative loci on chromosome 1p36.11-p35.1 and 4q28.1-q28.2. The latter locus (4q28.1-q28.2) contained the MFSD8 gene, comprising a novel homozygous missense mutation in exon 5 (c.362a>g /p.Tyr121Cys), which segregated with the disease in the three affected sibs. We describe a novel mutation in the previously identified MFSD8 gene in a family with a common phenotype of LINCL, but no clinical report of vision loss. Our results enlarge the mutational and perhaps the nosological spectrum of one of the recently identified subtypes of NCL, called CLN7.

Idiopathic generalized epilepsy phenotypes associated with different EFHC1 mutations.

We sequenced 61 patients with various idiopathic generalized epilepsy (IGE) syndromes for mutations in the EFHC1 gene. We detected three novel heterozygous missense mutations (I174V, C259Y, A394S) and one possibly pathogenic variant in the 3' UTR (2014t>c). The mutation I174V was also detected in 1 of 372 screened patients with temporal lobe epilepsy. We conclude that mutations in the EFHC1 gene may underlie different types of epilepsy syndromes.

Mutations in the CLCN2 gene are a rare cause of idiopathic generalized epilepsy syndromes.

Mutations in the chloride channel gene CLCN2 have been reported in families with generalized and focal epilepsy syndromes. To evaluate the contribution of mutations in the CLCN2 gene to the etiology of epilepsies in our population, we screened 96 patients with different epilepsy syndromes and a putative genetic background. No definite mutations were found in our study population. We conclude that mutations in the CLCN2 gene are only a rare cause of idiopathic generalized epilepsy.

The sepiapterin reductase gene region reveals association in the PARK3 locus: analysis of familial and sporadic Parkinson's disease in European populations.

BACKGROUND: Parkinson's disease is a genetically complex disease with mixed mode of inheritance. Recently, a haplotype across the sepiapterin reductase (SPR) gene, which is located in the PARK3 linkage region, was shown to modulate age of onset of Parkinson's disease in sibships from North America. OBJECTIVE: To make a thorough assessment of the SPR gene region in sporadic Parkinson's disease. METHODS: A linkage study in 122 European sibship families with five microsatellite and 17 single nucleotide polymorphism (SNP) markers in and around the SPR gene region, and an association analysis in 340 sporadic cases of Parkinson's disease and 680 control subjects from Germany with 40 SNPs. Linkage was evaluated by non-parametric linkage scores and genotypic or haplotype association was tested by regression analysis, assuming different risk effect models. RESULTS: Significant LOD scores between 2 and 3 were obtained at the two SPR-flanking markers D2S2110 and D2S1394 and seven SNP markers around the SPR gene. We found the previously reported promoter SNP rs1876487 also significantly associated with age of onset in our sib pair families (p-value 0.02). One strong linkage disequilibrium (LD) block of 45 kb including the entire SPR gene was observed. Within this LD block all 14 inter-correlated SNPs were significantly associated with Parkinson's disease affection status (p-value 0.004). CONCLUSIONS: DNA polymorphisms in a highly intercorrelated LD block, which includes the SPR gene, appear to be associated with both sporadic and familial Parkinson's disease. This confirms a previous study showing that SPR potentially modulates the onset of or risk for Parkinson's disease.

Inclusion body myopathy and Paget disease is linked to a novel mutation in the VCP gene.

Mutations in the valosin-containing protein (VCP) on chromosome 9p13-p12 were recently found to be associated with hereditary inclusion body myopathy, Paget disease of the bone, and frontotemporal dementia (IBMPFD). We identified a novel missense mutation in the VCP gene (R159H; 688G>A) segregating with this disease in an Austrian family of four affected siblings, who exhibited progressive proximal myopathy and Paget disease of the bone but without clinical signs of dementia.

A novel TITF-1 mutation causes benign hereditary chorea with response to levodopa.

Presented is a pedigree with infancy-onset benign hereditary chorea (BHC) caused by a novel nonsense mutation in exon 3 (523G-->T, E175X) of the TITF-1 (Nkx2.1) gene. Four confirmed mutation carriers showed the typical movement disorder of BHC and congenital hypothyroidism. Surprisingly, treatment with levodopa improved gait dramatically and reduced chorea in two patients. Dopaminergic drugs should be considered a useful therapeutic option in BHC.

The role of alpha-synuclein gene multiplications in early-onset Parkinson's disease and dementia with Lewy bodies.

BACKGROUND: A triplication of the alpha-synuclein gene was found to cause autosomal dominant Lewy body disease in two distinct families. METHOD: We searched for alterations of alpha-synuclein gene dosage and analysed the entire coding region for point mutations in 54 dementia with Lewy body disease (DLB) and in 103 young onset Parkinson's disease (PD) patients from Central Europe. RESULTS: We could not detect any quantitative alterations in the gene dosage of alpha-synuclein. Mutational screening of the entire coding region of alpha-synuclein revealed only one silent mutation V3V (adenine9guanine) in one case. CONCLUSIONS: Thus, this phenomenon appears not to be a major cause in the pathogenesis of sporadic DLB and young onset PD in this European population.

Genome-wide scan linkage analysis for Parkinson's disease: the European genetic study of Parkinson's disease.

OBJECTIVE: To undertake a full genome-wide screen for Parkinson's disease susceptibility loci. METHODS: A genome-wide linkage study was undertaken in 227 affected sibling pairs from 199 pedigrees with Parkinson's disease. The pedigree sample consisted of 188 pedigrees from five European countries, and 11 from the USA. Individuals were genotyped for 391 microsatellite markers at approximately 10 cM intervals throughout the genome. Multipoint model-free affected sibling pair linkage analyses were carried out using the MLS (maximum LOD score) test. RESULTS: There were six chromosomal regions with maximum MLS peaks of 1 or greater (pointwise p<0.018). Four of these chromosomal regions appear to be newly identified regions, and the highest MLS values were obtained on chromosomes 11q (MLS = 1.60, at 91 cM, D11S4175) and 7p (MLS = 1.51, at 5 cM, D7S531). The remaining two MLS peaks, on 2p11-q12 and 5q23, are consistent with excess sharing in regions reported by other studies. The highest MLS peak was observed on chromosome 2p11-q12 (MLS = 2.04, between markers D2S2216 and D2S160), within a relatively short distance (approximately 17 cM) from the PARK3 region. Although a stronger support of linkage to this region was observed in the late age of onset subgroup of families, these differences were not significant. The peak on 5q23 (MLS = 1.05, at 130 cM, D5S471) coincides with the region identified by three other genome scans. All peak locations fell within a 10 cM distance. CONCLUSIONS: These stratified linkage analyses suggest linkage heterogeneity within the sample across the 2p11-q12 and 5q23 regions, with these two regions contributing independently to Parkinson's disease susceptibility.

Association of an ABCB1 gene haplotype with pharmacoresistance in temporal lobe epilepsy.

The multidrug transporter P-glycoprotein is suspected of contributing to pharmacoresistance in temporal lobe epilepsy (TLE). To assess the role of functional variations in its coding gene (ABCB1) the authors genotyped 210 patients with TLE who were stratified according to their degree of drug resistance. They identified a common haplotype that when present in the homozygous state significantly increased the risk for pharmacoresistance.

Autosomal dominant parkinsonism associated with variable synuclein and tau pathology.

Since the original 1995 report of a parkinsonian kindred, four individuals have been affected (mean age at onset, 65 years). All four had cardinal signs of Parkinson disease (PD) and good response to levodopa. Four autopsies showed neuronal loss and gliosis in the substantia nigra. Lewy bodies (LB) limited to brainstem nuclei were detected in one case, diffuse LB in the second, neurofibrillary tangles (NFT) without LB in the third, and neither NFT nor LB in the fourth. Genetic studies suggested linkage to the PARK8 locus on chromosome 12.

Refinement of the PARK3 locus on chromosome 2p13 and the analysis of 14 candidate genes.

Parkinson's disease (PD) is a common neurodegenerative disorder with clinical features of bradykinesia, rigidity, resting tremor and postural instability resulting from the deficiency of dopamine in the nigrostriatal system. Previously we mapped a susceptibility gene for an autosomal dominant form of PD to a 10.6 cM region of chromosome 2p (PARK3; OMIM 602404). A common haplotype shared by two North American kindreds (Families B and C) genealogically traced to Southern Denmark and Northern Germany suggested a founder effect. Here we report progress in the refinement of the PARK3 locus and sequence analysis of candidate genes within the region. Members of families B and C were genotyped using polymorphic markers, reducing the minimum common haplotype to eight markers spanning a physical distance of 2.5 Mb. Analysis of 14 genes within the region did not reveal any potentially pathogenic mutations segregating with the disease, implying that none of these genes are likely candidates for PARK3.