Jelena Medic

Evidence for an oligogenic basis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with a substantial heritable component. In pedigrees affected by its familial form, incomplete penetrance is often observed. We hypothesized that this could be caused by a complex inheritance of risk variants in multiple genes. Therefore, we screened 111 familial ALS (FALS) patients from 97 families, and large cohorts of sporadic ALS (SALS) patients and control subjects for mutations in TAR DNA-binding protein (TARDBP), fused in sarcoma/translated in liposarcoma (FUS/TLS), superoxide dismutase-1 (SOD1), angiogenin (ANG) and chromosome 9 open reading frame 72 (C9orf72). Mutations were identified in 48% of FALS families, 8% of SALS patients and 0.5% of control subjects. In five of the FALS families, we identified multiple mutations in ALS-associated genes. We detected FUS/TLS and TARDBP mutations in combination with ANG mutations, and C9orf72 repeat expansions with TARDBP, SOD1 and FUS/TLS mutations. Statistical analysis demonstrated that the presence of multiple mutations in FALS is in excess of what is to be expected by chance (P = 1.57 × 10(-7)). The most compelling evidence for an oligogenic basis was found in individuals with a p.N352S mutation in TARDBP, detected in five FALS families and three apparently SALS patients. Genealogical and haplotype analyses revealed that these individuals shared a common ancestor. We obtained DNA of 14 patients with this TARDBP mutation, 50% of whom had an additional mutation (ANG, C9orf72 or homozygous TARDBP). Hereby, we provide evidence for an oligogenic aetiology of ALS. This may have important implications for the interpretation of whole exome/genome experiments designed to identify new ALS-associated genes and for genetic counselling, especially of unaffected family members.

Hexanucleotide repeat expansions in C9ORF72 in the spectrum of motor neuron diseases

Objective: To assess the frequency and phenotype of hexanucleotide repeat expansions in C9ORF72 in a large cohort of patients of Dutch descent with familial (fALS) and sporadic (sALS) amyotrophic lateral sclerosis (ALS), progressive muscular atrophy (PMA), and primary lateral sclerosis (PLS). Methods: Included were 78 patients with fALS, 1,422 with sALS, 246 with PMA, and 110 with PLS, and 768 control subjects. Repeat expansions were determined by a repeat primed PCR. Familial aggregation of dementia and Parkinson disease (PD) was examined among patients with ALS who carried the repeat expansion. Results: The expanded repeat was found in 33 (37%) of all patients with fALS, in 87 (6.1%) patients with sALS, in 4 (1.6%) patients with PMA, and in 1 (0.9%) patient with PLS. None of the controls carried the mutation. Patients with ALS with the repeat expansion had an earlier age at onset (median 59.3 vs 61.9 years, hazard ratio 1.55, p = 5 × 10−5) and shorter survival (median 2.5 vs 2.7 years, hazard ratio 1.46, p = 8 × 10−4). Dementia, but not PD, occurred nearly twice as often in relatives of patients with the expansion compared to all patients with ALS or controls (p = 9 × 10−4). Conclusions: The hexanucleotide repeat expansion in C9ORF72 is a major cause of fALS and apparently sporadic ALS in the Netherlands. Patients who carry the repeat expansion have an earlier onset, shorter survival, and familial aggregation of dementia. These results challenge the classic definition of fALS and may justify genetic testing in patients with sALS.

VAPB and C9orf72 mutations in 1 familial amyotrophic lateral sclerosis patient

Previously, we have reported amyotrophic lateral sclerosis (ALS) families with multiple mutations in major ALS-associated genes. These findings provided evidence for an oligogenic basis of ALS. In our present study, we screened a cohort of 755 sporadic ALS patients, 111 familial ALS patients (97 families), and 765 control subjects of Dutch descent for mutations in vesicle-associated membrane protein B (VAPB). We have identified 1 novel VAPB mutation (p.V234I) in a familial ALS patient known to have a chromosome 9 open reading frame 72 (C9orf72) repeat expansion. This p.V234I mutation was absent in control subjects, located in a region with high evolutionary conservation, and predicted to have damaging effects. Taken together, these findings provide additional evidence for an oligogenic basis of ALS.

Association of the TGF-beta receptor genes with abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a multifactorial condition. The transforming growth factor β (TGF-β) pathway regulates vascular remodeling and mutations in its receptor genes, TGFBR1 and TGFBR2, cause syndromes with thoracic aortic aneurysm (TAA). The TGF-β pathway may be involved in aneurysm development in general. We performed an association study by analyzing all the common genetic variants in TGFBR1 and TGFBR2 using tag single nucleotide polymorphisms (SNPs) in a Dutch AAA case–control population in a two-stage genotyping approach. In stage 1, analyzing 376 cases and 648 controls, three of the four TGFBR1 SNPs and nine of the 28 TGFBR2 SNPs had a P<0.07. Genotyping of these SNPs in an independent cohort of 360 cases and 376 controls in stage 2 confirmed association (P<0.05) for the same allele of one SNP in TGFBR1 and two SNPs in TGFBR2. Joint analysis of the 736 cases and 1024 controls showed statistically significant associations of these SNPs, which sustained after proper correction for multiple testing (TGFBR1 rs1626340 OR 1.32 95% CI 1.11–1.56 P=0.001 and TGFBR2 rs1036095 OR 1.32 95% CI 1.12–1.54 P=0.001 and rs4522809 OR 1.28 95% CI 1.12–1.46 P=0.0004). We conclude that genetic variations in TGFBR1 and TGFBR2 associate with AAA in the Dutch population. This suggests that AAA may develop partly by similar defects as TAA, which in the future may provide novel therapeutic options.

Pharmacokinetics of intravenous immunoglobulin in multifocal motor neuropathy

Background Multifocal motor neuropathy (MMN) is often responsive to treatment with intravenous immunoglobulin (IVIg), but the optimal dose and intervals of IVIg maintenance treatment have not been established. Increase in IgG concentration (ΔIgG) after IVIg infusion has recently been identified as determinant of outcome in Guillain-Barré syndrome. ΔIgG may therefore represent a potentially useful biomarker to optimise IVIg dosing in patients with MMN. Objective The aims of this study were to determine variability of IVIg pharmacokinetics in patients with MMN in relation to treatment response, and to establish whether interindividual differences in IVIg pharmacokinetics were associated with genetic polymorphisms of the endothelial IgG receptor (FcRn) which determines IgG half-life. Methods Twenty-three patients with MMN receiving their first IVIg treatment at a cumulative dose of 2.0 g/kg in 5 days were included. A good treatment response was defined as an increase in muscle strength of at least one Medical Research Council point in minimally two muscle groups. IgG concentrations in serum were determined at baseline, at day 1 and day 5 of the IVIg course, and 3 weeks after treatment. FcRn copy number variation and differences in repeat length of the variable number of tandem repeats in the FcRn gene were determined by quantitative PCR and Sanger sequencing. Results Seventeen patients (74%) had a good response to treatment. Total IgG and ΔIgG levels showed large variation between patients. Mean ΔIgG was higher in IVIg responders than in non-responders, with the largest difference on day 1 (11.1 g/L vs 4.5 g/L, p=0.06), but our study lacked power to show statistically significant differences. Genetic variation in the FcRn gene was not associated with ΔIgG levels or response to treatment. Conclusions IVIg pharmacokinetics varies in patients with MMN and may be associated with clinical response.

Genomic DNA Pooling Strategy for Next-Generation Sequencing-Based Rare Variant Discovery in Abdominal Aortic Aneurysm Regions of Interest—Challenges and Limitations

The costs and efforts for sample preparation of hundreds of individuals, their genomic enrichment for regions of interest, and sufficient deep sequencing bring a significant burden to next-generation sequencing-based experiments. We investigated whether pooling of samples at the level of genomic DNA would be a more versatile strategy for lowering the costs and efforts for common disease-associated rare variant detection in candidate genes or associated loci in a substantial patient cohort. We performed a pilot experiment using five pools of 20 abdominal aortic aneurysm (AAA) patients that were enriched on separate microarrays for the reported 9p21.3 associated locus and 42 additional AAA candidate genes, and sequenced on the SOLiD platform. Here, we discuss challenges and limitations connected to this approach and show that the high number of novel variants detected per pool and allele frequency deviations to the usually highly false positive cut-off region for variant detection in non-pooled samples can be limiting factors for successful variant prioritization and confirmation. We conclude that barcode indexing of individual samples before pooling followed by a multiplexed enrichment strategy should be preferred for detection of rare genetic variants in larger sample sets rather than a genomic DNA pooling strategy.

Incomplete segregation of MYH11 variants with thoracic aortic aneurysms and dissections and patent ductus arteriosus

Thoracic aortic aneurysms and dissections (TAAD) is a serious condition with high morbidity and mortality. It is estimated that 20% of non-syndromic TAAD cases are inherited in an autosomal-dominant pattern with variable expression and reduced penetrance. Mutations in myosin heavy chain 11 (MYH11), one of several identified TAAD genes, were shown to simultaneously cause TAAD and patent ductus arteriosus (PDA). We identified two large Dutch families with TAAD/PDA and detected two different novel heterozygote MYH11 variants in the probands. These variants, a heterozygote missense variant and a heterozygote in-frame deletion, were predicted to have damaging effects on protein structure and function. However, these novel alterations did not segregate with the TAAD/PDA in 3 out of 11 cases in family TAAD01 and in 2 out of 6 cases of family TAAD02. No mutation was detected in other known TAAD genes. Thus, it is expected that within these families other genetic factors contribute to the disease either by themselves or by interacting with the MYH11 variants. Such an oligogenic model for TAAD would explain the variable onset and progression of the disorder and its reduced penetrance in general. We conclude that in familial TAAD/PDA with an MYH11 variant in the index case caution should be exercised upon counseling family members. Specialized surveillance should still be offered to the non-carriers to prevent catastrophic aortic dissections or ruptures. Furthermore, our study underscores that segregation analysis remains very important in clinical genetics. Prediction programs and mutation evaluation algorithms need to be interpreted with caution.

Association study of single nucleotide polymorphisms on chromosome 19q13 with abdominal aortic aneurysm

Background: Abdominal aortic aneurysm (AAA) is a complex disorder in which environmental and genetic factors play a role in pathogenesis. Linkage to 2 adjacent loci on 19q13 in familiar AAA was previously demonstrated. We studied whether genetic variation within these regions predisposes to AAA. Methods: Common genetic variants in the described regions on 19q13 were analyzed using tag single nucleotide polymorphisms (SNPs) in a Dutch case-control population. Single nucleotide polymorphism genotyping was performed in a 2-stage approach. Results: In stage 1, 615 SNPs were genotyped in 376 AAA patients and 648 controls. In stage 2, 8 SNPs of stage 1 with a P value < .015 were genotyped in a second independent cohort of 360 cases and 376 controls. No differences in allele frequencies were observed. Conclusion: Our findings suggest that there are no common AAA predisposing SNPs within the 19q13 loci. Hence, the genetic basis of familiar and sporadic AAA may differ.

Genes involved in the transforming growth factor beta signalling pathway and the risk of intracranial aneurysms

Background and purpose: The 19q13.3 locus for intracranial aneurysms (IA) partly overlaps with the 19q13 locus for abdominal aortic aneurysms (AAA). A common genetic risk factor located in this locus for the two aneurysm types seems plausible. The transforming growth factor beta (TGF-β) signalling pathway plays a role in aortic aneurysms but may also play a role in aneurysms in general. In the combined region of the 19q13 loci for IA and AAA we identified two candidate genes that are both involved in the TGF-β signalling pathway: hepsin (HPN) and the latent transforming growth factor beta-binding protein 4 (LTBP4). We hypothesised that single nucleotide polymorphisms (SNP) in the HPN and LTBP4 genes are associated with IA. Methods: We analyzed all the common variations using tag SNP in the HPN and LTBP4 genes for association with IA in 390 patients and 642 controls in the Dutch population. Six tag SNP in the HPN gene and five tag SNP in the LTBP4 gene were genotyped. Results: No differences in SNP frequency were observed for both the HPN and LTBP4 gene between patients and controls. Conclusion: Our findings suggest that variations in or near the HPN and LTBP4 genes do not play a role in the susceptibility to IA in the Dutch population.

The Intracranial Aneurysm Susceptibility Genes HSPG2 and CSPG2 Are Not Associated With Abdominal Aortic Aneurysm

Background: A genetic variant on chromosome 9p21 associates with abdominal aortic aneurysm (AAA) and intracranial aneurysm (IA), indicating that despite the differences in pathology there are shared genetic risk factors. We investigated whether the IA susceptibility genes heparan sulfate proteoglycan 2 (HSPG2) and chondroitin sulfate proteoglycan 2 (CSPG2) associate with AAA as well. Methods: Using tag single nucleotide polymorphisms (SNPs), all common variants were analyzed in a Dutch AAA case-control population in a 2-stage genotyping approach. In stage 1, 12 tag SNPs in HSPG2 and 22 tag SNPs in CSPG2 were genotyped in 376 patients and 648 controls. Genotyping of significantly associated SNPs was replicated in a second independent cohort of 360 cases and 376 controls. Results: In stage 1, no HSPG2 SNPs and 1 CSPG2 SNP associated with AAA (rs2652106, P = .019). Association of this SNP was not replicated (P = .342). Conclusions: Our findings demonstrate that, in contrast to IA, HSPG2 and CSPG2 do not associate with AAA.