Search

Next-generation sequencing (NGS) will soon be used for clinically heterogeneous, inherited disorders and the increasing number of disease-causing genes reported. Diagnostic laboratories therefore need to decide which NGS methods they are going to invest in and how to implement them. We discuss here the challenges and opportunities of using targeted resequencing (TRS) panels for diagnosing monogenetic disorders. Of the different NGS approaches available, TRS panels offer the opportunity to sequence and analyze a limited set of predetermined target genes. At present, TRS panels offer better base-pair coverage, running times, costs and dataset handling than other NGS applications such as whole genome sequencing and whole exome sequencing. However, working with TRS panels also poses new challenges in variant interpretation, data handling and bioinformatic analyses. To optimize the analyses, TRS panel testing should be performed by bioinformaticians, clinicians and laboratory staff in close collaboration. [ABSTRACT FROM AUTHOR]

ABSTRACT Background Ramsay Hunt syndrome (progressive myoclonus ataxia) is a descriptive diagnosis characterized by myoclonus, ataxia, and infrequent seizures. Often the etiology cannot be determined. Recently, a mutation in the GOSR2 gene (c.430G>T, p.Gly144Trp) was reported in 6 patients with childhood-onset progressive ataxia and myoclonus. Methods We evaluated 5 patients with cortical myoclonus, ataxia, and areflexia. Results All 5 patients had the same homozygous mutation in GOSR2. Here we present their clinical and neurophysiological data. Our patients (aged 7-26 years) all originated from the northern Netherlands and showed a remarkably homogeneous phenotype. Myoclonus and ataxia were relentlessly progressive over the years. Electromyography revealed signs of sensory neuronopathy or anterior horn cell involvement, or both, in all patients with absent reflexes. Conclusions Based on the presented phenotype, we would advise movement disorder specialists to consider mutation analysis of GOSR2 in patients with Ramsay Hunt syndrome, especially when they also have areflexia. © 2013 International Parkinson and Movement Disorder Society [ABSTRACT FROM AUTHOR]

The correct interpretation of copy number gains in patients with developmental delay and multiple congenital anomalies is hampered by the large number of copy number variations (CNVs) encountered in healthy individuals. The variable phenotype associated with copy number gains makes interpretation even more difficult. Literature shows that inheritence, size and presence in healthy individuals are commonly used to decide whether a certain copy number gain is pathogenic, but no general consensus has been established. We aimed to develop guidelines for interpreting gains detected by array analysis using array CGH data of 300 patients analysed with the 105K Agilent oligo array in a diagnostic setting. We evaluated the guidelines in a second, independent, cohort of 300 patients. In the first 300 patients 797 gains of four or more adjacent oligonucleotides were observed. Of these, 45.4% were de novo and 54.6% were familial. In total, 94.8% of all de novo gains and 87.1% of all familial gains were concluded to be benign CNVs. Clinically relevant gains ranged from 288 to 7912 kb in size, and were significantly larger than benign gains and gains of unknown clinical relevance (P<0.001). Our study showed that a threshold of 200 kb is acceptable in a clinical setting, whereas heritability does not exclude a pathogenic nature of a gain. Evaluation of the guidelines in the second cohort of 300 patients revealed that the interpretation guidelines were clear, easy to follow and efficient. [ABSTRACT FROM AUTHOR]

We present a linkage study in a four-generation autosomal dominant cerebellar ataxia (ADCA) family of Dutch ancestry. The family shows a clinically and genetically distinct form of ADCA. This neurodegenerative disorder manifests in the family as a relatively mild ataxia syndrome with some additional characteristic symptoms. We have identified a SCA19 locus, approved by the Human Genome Nomenclature Committee that can be assigned to the chromosome region 1p21-q21. Our mutation analysis failed to identify any mutations in the known spinocerebellar ataxia (SCA) genes and linkage analysis excluded the remaining SCA loci. We therefore performed a genome-wide scan with 350 microsatellite markers to identify the location of the disease-causing gene in this family. Multi-point analysis was performed and exclusion maps were generated. Linkage and haplotype analysis revealed linkage to an interval located on chromosome 1. The estimated minimal prevalence of ADCA in the Netherlands is about 3:100,000. To date, sixteen different SCA loci have been identified in ADCA (SCA1–8 and SCA10–17). However, mutation analysis has been commercially available only for the SCA1, 2, 3, 6 and 7 genes. So far, a molecular analysis in these SCA genes cannot be made in about one-third of the ADCA families. Thus, the identification of this new, additional SCA19 locus will contribute to expanding the DNA diagnostic possibilities. [ABSTRACT FROM AUTHOR]

The article offers information on the genetic aspects of dilated cardiomyopathy (CMD). Idiopathic dilated cardiomyopathy (IDC) or CMD is defined by the presence of left ventricular dilatation and systolic dysfunction in the absence of an underlying cause, such as hypertension, valve disease or coronary artery disease. Research has revealed 51 DCM genes and majority of the mutations in these genes are missense mutations.

Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disorder caused by mutations in the neuronal-specific protein kinase C gamma (PKC γ) gene. Since most mutations causing SCA14 are located in the PKC γ C1B regulatory subdomain, we investigated the impact of three C1B mutations on the intracellular kinetics, protein conformation and kinase activity of PKC γ in living cells. SCA14 mutant PKC γ proteins showed enhanced phorbol-ester-induced kinetics when compared with wild-type PKC γ. The mutations led to a decrease in intramolecular FRET of PKC γ, suggesting that they 'open' PKC γ protein conformation leading to unmasking of the phorbol ester binding site in the C1 domain. Surprisingly, SCA14 mutant PKC γ showed reduced kinase activity as measured by phosphorylation of PKC γ reporter MyrPalm-CKAR, as well as downstream components of the MAPK signaling pathway. Together, these results show that SCA14 mutations located in the C1B subdomain 'open' PKC γ protein conformation leading to increased C1 domain accessibility, but inefficient activation of downstream signaling pathways. [ABSTRACT FROM AUTHOR]

Background: Genetic disorders causing dystonia show great heterogeneity. Recent studies have suggested that next-generation sequencing techniques such as gene panel analysis can be effective in diagnosing heterogeneous conditions. The objective of this study was to investigate whether dystonia patients with a suspected genetic cause could benefit from the use of gene panel analysis., Methods: In this post hoc study, we describe gene panel analysis results of 61 dystonia patients (mean age, 31 years; 72% young onset) in our tertiary referral center. The panel covered 94 dystonia-associated genes. As comparison with a historic cohort was not possible because of the rapidly growing list of dystonia genes, we compared the diagnostic workup with and without gene panel analysis in the same patients. The workup without gene panel analysis (control group) included theoretical diagnostic strategies formulated by independent experts in the field, based on detailed case descriptions. The primary outcome measure was diagnostic yield; secondary measures were cost and duration of diagnostic workup., Results: Workup with gene panel analysis led to a confirmed molecular diagnosis in 14.8%, versus 7.4% in the control group (P = 0.096). In the control group, on average 3 genes/case were requested. The mean costs were lower in the gene panel analysis group (€1822/case) than in the controls (€2660/case). The duration of the workup was considerably shorter with gene panel analysis (28 vs 102 days)., Conclusions: Gene panel analysis facilitates molecular diagnosis in complex cases of dystonia, with a good diagnostic yield (14.8%), a quicker diagnostic workup, and lower costs, representing a major improvement for patients and their families. © 2016 International Parkinson and Movement Disorder Society., (© 2017 International Parkinson and Movement Disorder Society.)

Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disorder caused by mutations in the neuronal-specific protein kinase C gamma (PKCgamma) gene. Since most mutations causing SCA14 are located in the PKCgamma C1B regulatory subdomain, we investigated the impact of three C1B mutations on the intracellular kinetics, protein conformation and kinase activity of PKCgamma in living cells. SCA14 mutant PKCgamma proteins showed enhanced phorbol-ester-induced kinetics when compared with wild-type PKCgamma. The mutations led to a decrease in intramolecular FRET of PKCgamma, suggesting that they ;open' PKCgamma protein conformation leading to unmasking of the phorbol ester binding site in the C1 domain. Surprisingly, SCA14 mutant PKCgamma showed reduced kinase activity as measured by phosphorylation of PKC reporter MyrPalm-CKAR, as well as downstream components of the MAPK signaling pathway. Together, these results show that SCA14 mutations located in the C1B subdomain ;open' PKCgamma protein conformation leading to increased C1 domain accessibility, but inefficient activation of downstream signaling pathways.

Background: Evidence is emerging for the association of aberrant homocysteine-methylation cycle and increased risk of schizophrenia., Methods: We examined the prevalence of the catechol-O-methyltransferase (COMT) 324G>A (Val108/158Met) and methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphisms in 252 patients with schizophrenia and 405 control subjects. All subjects were of Dutch ancestry., Results: The COMT 324AA genotype was not associated with an increased risk of schizophrenia (odds ratio (OR)=1.38 [95% CI: 0.88-2.16], P=0.162), and the MTHFR 677TT genotype showed a nearly significant increased risk for schizophrenia (OR=1.65 [95% CI: 0.97-2.82], P=0.067). The odds ratio for schizophrenia associated with joint occurrence of the COMT 324AA and MTHFR 677TT genotype was 3.08 (95% CI: 1.08-8.76) (P=0.035). Increasing number of low enzyme activity alleles in the COMT and MTHFR genotype combinations were associated with an increased risk of schizophrenia (test for trend, P=0.017)., Conclusions: Our findings do not support a major role for the COMT 324AA and MTHFR 677TT genotype alone, but the combination of both genotypes might increase schizophrenia susceptibility.

X-linked agammaglobulinemia (XLA) is a primary immunodeficiency disorder caused by germline mutation of the Bruton tyrosine kinase (BTK) gene. It is characterized by disturbed B-cell development, decreased immunoglobulin levels, and increased patient susceptibility to infection. An increased risk of cancer has been suggested, but most reports were described before the identification of BTK gene mutation as the cause of XLA. Here we describe 2 patients with genetically ascertained XLA and multiple colorectal neoplasms, supporting an increased risk of colorectal cancer in XLA and highlighting the potential importance of colorectal surveillance in these patients.

Joubert syndrome (JBS) is a clinically variable and genetically heterogeneous developmental brain disorder with autosomal recessive inheritance. Five genes, AHI1, NPHP1, CEP290, MKS3, and RPGRIP1L, and two additional loci on chromosome 9 and 11 have been identified so far. The relative contributions of AHI1 mutations and NPHP1 deletions have not yet been determined in a population-based JBS patient cohort. We therefore undertook a nationwide survey of JBS in the Netherlands and performed DNA analysis of the AHI1 and NPHP1 genes, as well as a new candidate gene CYCLIN D1. We obtained clinical data and DNA samples of 25 Dutch JBS patients. DNA analysis of AHI1 revealed pathogenic homozygous or compound heterozygous AHI1 mutations in four patients (16%). Based on the birth prevalence of about 1 in 100,000 for JBS in the Netherlands, we estimated a carrier frequency of AHI1 mutations of approximately 1 in 400. In another two patients, the AHI1 mutation Arg830Trp was identified (homozygously and heterozygously), a possible low penetrance allele. No deletions of NPHP1 or CYCLIN D1 mutations were detected in these 25 patients. In the four patients with AHI1 mutations, retinal disease (Leber congenital amaurosis or retinal dystrophy) was present in two, whereas none had renal disease. Pooling our data and data from the literature, retinal disease seems to occur in 75% of AHI1-associated JBS patients. Renal disease is present in 10% at most. We conclude that AHI1 mutations are an important cause of JBS in Dutch patients, and should always be looked for in patients suspected of JBS, especially when retinal dystrophy is present. Patients with AHI1 mutations should be regularly checked for retinal and renal disease up until adolescence.

In dominant spinocerebellar ataxias (SCAs), the issue of whether non-CAG dependent factors contribute to onset age remains unsettled. Data on SCA genotype, onset age, normal/expanded CAG repeat length, sex of the patient and transmitting parent, and family details were available from 802 patients. Based on the model [log(10) (age at onset) = k - b CAG(exp) + epsilon], we examined changes in adjusted R(2) and residual standard error following incorporation of the other factors in this model. The expanded repeat explained 44.3 to 74.9% of onset age variance, although this was less than 50% in SCA3 and SCA6, implicating a large effect of non-CAG factors. The relation between onset age and CAG repeat was similar for SCA1, 3, 6, and 7, but different for SCA2, pointing to different polyglutamine effects in SCA2. For SCA2 and SCA3, 17.1 and 45.5% of onset age variance, respectively, were explained by currently (unidentified) familial factors. We found a significant contribution of the nonexpanded allele in SCA1 and SCA6. Besides polyglutamine motif (determined by the expanded CAG repeat length), we identified the following age at onset modifiers: protein context in SCA2; familial factors in SCA2 and SCA3; and the nonexpanded CAG repeat in SCA1 and SCA6.

In recent years, molecular genetic research has unraveled a major part of the genetic background of autosomal dominant and recessive spinocerebellar ataxias. These advances have also allowed insight in (some of) the pathophysiologic pathways assumed to be involved in these diseases. For the clinician, the expanding number of genes and genetic loci in these diseases and the enormous clinical heterogeneity of specific ataxia subtypes complicate management of ataxia patients. In this review, the clinical and neuropathologic features of the recently identified spinocerebellar ataxias are described, and the various molecular mechanisms that have been demonstrated to be involved in these disorders are discussed.