Eric A.M. Hennekam

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.

Mutations in CCBE1 cause generalized lymph vessel dysplasia in humans

Lymphedema, lymphangiectasias, mental retardation and unusual facial characteristics define the autosomal recessive Hennekam syndrome. Homozygosity mapping identified a critical chromosomal region containing CCBE1, the human ortholog of a gene essential for lymphangiogenesis in zebrafish. Homozygous and compound heterozygous mutations in seven subjects paired with functional analysis in a zebrafish model identify CCBE1 as one of few genes causing primary generalized lymph-vessel dysplasia in humans.

Mutations in WNT10A are present in more than half of isolated hypodontia cases

Background Dental agenesis is the most common, often heritable, developmental anomaly in humans. Mutations in MSX1, PAX9, AXIN2 and the ectodermal dysplasia genes EDA, EDAR and EDARADD have been detected in familial severe tooth agenesis. However, until recently, in the majority of cases (∼90%) the genetic factor could not be identified, implying that other genes must be involved. Recent insights into the role of Wnt10A in tooth development, and the finding of hypodontia in carriers of the autosomal recessive disorder, odontooncychodermal dysplasia, due to mutations in WNT10A (OMIM 257980; OODD), make WNT10A an interesting candidate gene for dental agenesis. Methods In a panel of 34 patients with isolated hypodontia, the candidate gene WNT10A and the genes MSX1, PAX9, IRF6 and AXIN2 have been sequenced. The probands all had isolated agenesis of between six and 28 teeth. Results WNT10A mutations were identified in 56% of the cases with non-syndromic hypodontia. MSX1, PAX9 and AXIN2 mutations were present in 3%, 9% and 3% of the cases, respectively. Conclusion The authors identified WNT10A as a major gene in the aetiology of isolated hypodontia. By including WNT10A in the DNA diagnostics of isolated tooth agenesis, the yield of molecular testing in this condition was significantly increased from 15% to 71%.

Angiogenin variants in Parkinson disease and amyotrophic lateral sclerosis

Several studies have suggested an increased frequency of variants in the gene encoding angiogenin (ANG) in patients with amyotrophic lateral sclerosis (ALS). Interestingly, a few ALS patients carrying ANG variants also showed signs of Parkinson disease (PD). Furthermore, relatives of ALS patients have an increased risk to develop PD, and the prevalence of concomitant motor neuron disease in PD is higher than expected based on chance occurrence. We therefore investigated whether ANG variants could predispose to both ALS and PD.

A CASE OF ALS-FTD IN A LARGE FALS PEDIGREE WITH A K17I ANG MUTATION

Approximately 90% of amyotrophic lateral sclerosis (ALS) cases are sporadic (SALS), but 10% are familial (FALS). Mutations in SOD1, Alsin , Dynactin , SETX , DJ-1 , VAPB , and TDP-431 have been reported (table e-1 on the Neurology ® Web site at www.neurology.org). After the identification of sequence variation VEGF in patients with ALS, mutations in another angiogenic gene ( ANG ) were identified in SALS and FALS.2,3 Studies in other populations have identified ANG mutations in patients with ALS, but also in healthy controls. This suggests that not all mutations are pathogenic.3,4 ### Methods. A total of 39 unrelated FALS patients, negative for SOD1 mutations, were screened for ANG mutations. This study was approved by the local ethics committee and participants provided informed consent. DNA was isolated from venous blood and ANG mutation analysis was performed as described in appendix e-1. A total of 275 unrelated, healthy controls were taken from a prospective population-based study on ALS in The Netherlands and were also screened.5 PMut (http://mmb2.pcb.ub.es:8080/PMut/) was used to predict the impact of an amino acid substitution on the structure and function of the protein. ### Results. We identified one mutation in one patient (122 A>T) (figure, A), leading to an amino acid substitution of lysine to isoleucine (K17I) (figure, B). PMut analysis predicted this mutation to be pathogenic. Sequence alignments of ANG in different species …

Paternal age and psychiatric disorders: findings from a Dutch population registry.

BACKGROUND We measured the association between paternal age and schizophrenia (SCZ), autism spectrum disorders (ASD), major depressive disorder (MDD), and bipolar disorder (BPD) in the Dutch population. METHODS In total, 14231 patients and 56924 matched controls were collected and analyzed for an association with paternal age by logistic regression. RESULTS ASD is significantly associated with increased paternal age: Older fathers >40 years of age have a 3.3 times increased odds of having a child with ASD compared to young fathers <20 years of age. SCZ has significant associations for fathers aged >35 years (OR=1.27, 95% Confidence Interval: 1.05 and 1.53). For MDD, both younger and older fathers have increased odds. No association was found for BPD. CONCLUSIONS The effects of paternal age as a risk factor are different for ASD and SCZ on one hand, and the affective disorders on the other hand. Different types of association might indicate different biological or psychosocial mechanisms. Late paternity (associated with predispositions to psychiatric disorders) seems the most probable explanation for the association with paternal age.

A double hit implicates DIAPH3 as an autism risk gene

Recent studies have shown that more than 10% of autism cases are caused by de novo structural genomic rearrangements. Given that some heritable copy number variants (CNVs) have been observed in patients as well as in healthy controls, to date little attention has been paid to the potential function of these non-de novo CNVs in causing autism. A normally intelligent patient with autism, with non-affected parents, was identified with a maternally inherited 10 Mb deletion at 13q21.2. Sequencing of the genes within the deletion identified a paternally inherited nonsynonymous amino-acid substitution at position 614 of diaphanous homolog 3 (DIAPH3) (proline to threonine; Pro614Thr). This variant, present in a highly conserved domain, was not found in 328 healthy subjects. Experiments showed a transient expression of Diaph3 in the developing murine cerebral cortex, indicating it has a function in brain development. Transfection of Pro614Thr in murine fibroblasts showed a significant reduction in the number of induced filopodia in comparison to the wild-type gene. DIAPH3 is involved in cell migration, axon guidance and neuritogenesis, and is suggested to function downstream of SHANK3. Our findings strongly suggest DIAPH3 as a novel autism susceptibility gene. Moreover, this report of a ‘double-hit’ compound heterozygote for a large, maternally inherited, genomic deletion and a paternally inherited rare missense mutation shows that not only de novo genomic variants in patients should be taken seriously in further study but that inherited CNVs may also provide valuable information.

Genetic Overlap between Apparently Sporadic Motor Neuron Diseases

Progressive muscular atrophy (PMA) and amyotrophic lateral sclerosis (ALS) are devastating motor neuron diseases (MNDs), which result in muscle weakness and/or spasticity. We compared mutation frequencies in genes known to be associated with MNDs between patients with apparently sporadic PMA and ALS. A total of 261 patients with adult-onset sporadic PMA, patients with sporadic ALS, and control subjects of Dutch descent were obtained at national referral centers for neuromuscular diseases in The Netherlands. Sanger sequencing was used to screen these subjects for mutations in the coding regions of superoxide dismutase-1 (SOD1), angiogenin (ANG), fused in sarcoma/translated in liposarcoma (FUS/TLS), TAR DNA-binding protein 43 (TARDBP), and multivesicular body protein 2B (CHMP2B). In our cohort of PMA patients we identified two SOD1 mutations (p.D90A, p.I113T), one ANG mutation (p.K17I), one FUS/TLS mutation (p.R521H), one TARDBP mutation (p.N352S), and one novel CHMP2B mutation (p.R69Q). The mutation frequency of these genes was similar in sporadic PMA (2.7%) and ALS (2.0%) patients, and therefore, our findings demonstrate a genetic overlap between apparently sporadic PMA and ALS.

A novel pathogenic MLH1 missense mutation, c.112A > C, p.Asn38His, in six families with Lynch syndrome

BackgroundAn unclassified variant (UV) in exon 1 of the MLH1 gene, c.112A > C, p.Asn38His, was found in six families who meet diagnostic criteria for Lynch syndrome. The pathogenicity of this variant was unknown. We aim to elucidate the pathogenicity of this MLH1 variant in order to counsel these families adequately and to enable predictive testing in healthy at-risk relatives.MethodsWe studied clinical data, microsatellite instability and immunohistochemical staining of MMR proteins, and performed genealogy, haplotype analysis and DNA testing of control samples.ResultsThe UV showed co-segregation with the disease in all families. All investigated tumors showed a microsatellite instable pattern. Immunohistochemical data were variable among tested tumors. Three families had a common ancestor and all families originated from the same geographical area in The Netherlands. Haplotype analysis showed a common haplotype in all six families.ConclusionsWe conclude that the MLH1 variant is a pathogenic mutation and genealogy and haplotype analysis results strongly suggest that it is a Dutch founder mutation. Our findings imply that predictive testing can be offered to healthy family members. The immunohistochemical data of MMR protein expression show that interpreting these results in case of a missense mutation should be done with caution.

Destabilized SMC5/6 complex leads to chromosome breakage syndrome with severe lung disease

The structural maintenance of chromosomes (SMC) family of proteins supports mitotic proliferation, meiosis, and DNA repair to control genomic stability. Impairments in chromosome maintenance are linked to rare chromosome breakage disorders. Here, we have identified a chromosome breakage syndrome associated with severe lung disease in early childhood. Four children from two unrelated kindreds died of severe pulmonary disease during infancy following viral pneumonia with evidence of combined T and B cell immunodeficiency. Whole exome sequencing revealed biallelic missense mutations in the NSMCE3 (also known as NDNL2) gene, which encodes a subunit of the SMC5/6 complex that is essential for DNA damage response and chromosome segregation. The NSMCE3 mutations disrupted interactions within the SMC5/6 complex, leading to destabilization of the complex. Patient cells showed chromosome rearrangements, micronuclei, sensitivity to replication stress and DNA damage, and defective homologous recombination. This work associates missense mutations in NSMCE3 with an autosomal recessive chromosome breakage syndrome that leads to defective T and B cell function and acute respiratory distress syndrome in early childhood.