Patrick J. Willems

Fmr1 knockout mice: A model to study fragile X mental retardation

Male patients with fragile X syndrome lack FMR1 protein due to silencing of the FMR1 gene by amplification of a CGG repeat and subsequent methylation of the promoter region. The absence of FMR1 protein leads to mental retardation, aberrant behavior, and macroorchidism. Hardly anything is known about the physiological function of FMR1 and the pathological mechanisms leading to these symptoms. Therefore, we designed a knockout model for the fragile X syndrome in mice. The knockout mice lack normal Fmr1 protein and show macroorchidism, learning deficits, and hyperactivity. Consequently, this knockout mouse may serve as a valuable tool in the elucidation of the physiological role of FMR1 and the mechanisms involved in macroorchidism, abnormal behavior, and mental retardation.

Abnormal dendritic spine characteristics in the temporal and visual cortices of patients with fragile‐X syndrome: A quantitative examination

Fragile-X syndrome is a common form of mental retardation resulting from the inability to produce the fragile-X mental retardation protein. Qualitative examination of human brain autopsy material has shown that fragile-X patients exhibit abnormal dendritic spine lengths and shapes on parieto-occipital neocortical pyramidal cells. Similar quantitative results have been obtained in fragile-X knockout mice, that have been engineered to lack the fragile-X mental retardation protein. Dendritic spines on layer V pyramidal cells of human temporal and visual cortices stained using the Golgi-Kopsch method were investigated. Quantitative analysis of dendritic spine length, morphology, and number was carried out on patients with fragile-X syndrome and normal age-matched controls. Fragile-X patients exhibited significantly more long dendritic spines and fewer short dendritic spines than did control subjects in both temporal and visual cortical areas. Similarly, fragile-X patients exhibited significantly more dendritic spines with an immature morphology and fewer with a more mature type morphology in both cortical areas. In addition, fragile-X patients had a higher density of dendritic spines than did controls on distal segments of apical and basilar dendrites in both cortical areas. Long dendritic spines with immature morphologies and elevated spine numbers are characteristic of early development or a lack of sensory experience. The fact that these characteristics are found in fragile-X patients throughout multiple cortical areas may suggest a global failure of normal dendritic spine maturation and or pruning during development that persists throughout adulthood.

A Point mutation in the FMR-1 gene associated with fragile X mental retardation.

The vast majority of patients with fragile X syndrome show a folate–sensitive fragile site at Xq27.3 (FRAXA) at the cytogenetic level, and both amplification of the (CGG)n repeat and hypermethylation of the CpG island in the 5′ fragile X gene (FMR–1) at the molecular level. We have studied the FMR–1 gene of a patient with the fragile X phenotype but without cytogenetic expression of FRAXA, a (CGG)n repeat of normal length and an unmethylated CpG island. We find a single point mutation in FMR–1 resulting in an Ne367Asn substitution. This de novo mutation is absent in the patients family and in 130 control X chromosomes, suggesting that the mutation causes the clinical abnormalities. Our results suggest that mutations in FMR–1 are directly responsible for fragile X syndrome, irrespective of possible secondary effects caused by FRAXA

Mutations in SMAD3 cause a syndromic form of aortic aneurysms and dissections with early-onset osteoarthritis

Thoracic aortic aneurysms and dissections are a main feature of connective tissue disorders, such as Marfan syndrome and Loeys-Dietz syndrome. We delineated a new syndrome presenting with aneurysms, dissections and tortuosity throughout the arterial tree in association with mild craniofacial features and skeletal and cutaneous anomalies. In contrast with other aneurysm syndromes, most of these affected individuals presented with early-onset osteoarthritis. We mapped the genetic locus to chromosome 15q22.2–24.2 and show that the disease is caused by mutations in SMAD3. This gene encodes a member of the TGF-β pathway that is essential for TGF-β signal transmission. SMAD3 mutations lead to increased aortic expression of several key players in the TGF-β pathway, including SMAD3. Molecular diagnosis will allow early and reliable identification of cases and relatives at risk for major cardiovascular complications. Our findings endorse the TGF-β pathway as the primary pharmacological target for the development of new treatments for aortic aneurysms and osteoarthritis.

HDAC8 mutations in Cornelia de Lange syndrome affect the cohesin acetylation cycle.

Cornelia de Lange syndrome (CdLS) is a dominantly inherited congenital malformation disorder, caused by mutations in the cohesin-loading protein NIPBL for nearly 60% of individuals with classical CdLS, and by mutations in the core cohesin components SMC1A (∼5%) and SMC3 (<1%) for a smaller fraction of probands. In humans, the multisubunit complex cohesin is made up of SMC1, SMC3, RAD21 and a STAG protein. These form a ring structure that is proposed to encircle sister chromatids to mediate sister chromatid cohesion and also has key roles in gene regulation. SMC3 is acetylated during S-phase to establish cohesiveness of chromatin-loaded cohesin, and in yeast, the class I histone deacetylase Hos1 deacetylates SMC3 during anaphase. Here we identify HDAC8 as the vertebrate SMC3 deacetylase, as well as loss-of-function HDAC8 mutations in six CdLS probands. Loss of HDAC8 activity results in increased SMC3 acetylation and inefficient dissolution of the ‘used’ cohesin complex released from chromatin in both prophase and anaphase. SMC3 with retained acetylation is loaded onto chromatin, and chromatin immunoprecipitation sequencing analysis demonstrates decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations.

Mutations in the facilitative glucose transporter GLUT10 alter angiogenesis and cause arterial tortuosity syndrome

Arterial tortuosity syndrome (ATS) is an autosomal recessive disorder characterized by tortuosity, elongation, stenosis and aneurysm formation in the major arteries owing to disruption of elastic fibers in the medial layer of the arterial wall. Previously, we used homozygosity mapping to map a candidate locus in a 4.1-Mb region on chromosome 20q13.1 (ref. 2). Here, we narrowed the candidate region to 1.2 Mb containing seven genes. Mutations in one of these genes, SLC2A10, encoding the facilitative glucose transporter GLUT10, were identified in six ATS families. GLUT10 deficiency is associated with upregulation of the TGFβ pathway in the arterial wall, a finding also observed in Loeys-Dietz syndrome, in which aortic aneurysms associate with arterial tortuosity. The identification of a glucose transporter gene responsible for altered arterial morphogenesis is notable in light of the previously suggested link between GLUT10 and type 2 diabetes. Our data could provide new insight on the mechanisms causing microangiopathic changes associated with diabetes and suggest that therapeutic compounds intervening with TGFβ signaling represent a new treatment strategy.

CRASH syndrome: clinical spectrum of corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraparesis and hydrocephalus due to mutations in one single gene, L1.

L1 is a neuronal cell adhesion molecule with important functions in the development of the nervous system. The gene encoding L1 is located near the telomere of the long arm of the X chromosome in Xq28. We review here the evidence that several X-linked mental retardation syndromes including X-linked hydrocephalus (HSAS), MASA syndrome, X-linked complicated spastic paraparesis (SP1) and X-linked corpus callosum agenesis (ACC) are all due to mutations in the L1 gene. The inter- and intrafamilial variability in families with an L1 mutation is very wide, and patients with HSAS, MASA, SP1 and ACC can be present within the same family. Therefore, we propose here to refer to this clinical syndrome with the acronym CRASH, for Corpus callosum hypoplasia, Retardation, Adducted thumbs, Spastic paraplegia and Hydrocephalus.

Mutations in the cell adhesion molecule LI cause mental retardation

Recently, studies in the usually disparate fields of human genetics and developmental neurobiology have converged to reveal that some types of human mental retardation and brain malformations are due to mutations that affect the neural cell adhesion molecule L1. L1 has a very complex biology, interacting with a variety of ligands, and functioning in migration of neurons and growth of axons. Over the past few years, it has also become clear that L1 is able to influence intracellular second messengers. The identification of a number of different mutations in L1, some of which alter the extracellular portion of the molecule, and others that change only the cytoplasmic tail, confirm that L1 is a crucial player in normal brain development. The information gained from genetic analysis of human L1 is giving new insights into how L1 functions in the formation of major axon pathways, but it also raises unanticipated questions about how L1 participates in the development of cortical and ventricular systems.

Transgenic mouse model for the fragile X syndrome

Transgenic fragile X knockout mice have been constructed to provide an animal model to study the physiologic function of the fragile X gene (FMR1) and to gain more insight into the clinical phenotype caused by the absence of the fragile X protein. Initial experiments suggested that the knockout mice show macroorchidism and cognitive and behavioral deficits, abnormalities comparable to those of human fragile X patients. In the present study, we have extended our experiments, and conclude that the Fmr1 knockout mouse is a reliable transgenic model to study the fragile X syndrome.

Phenotypic spectrum of the SMAD3-related aneurysms–osteoarthritis syndrome

Background Aneurysms–osteoarthritis syndrome (AOS) is a new autosomal dominant syndromic form of thoracic aortic aneurysms and dissections characterised by the presence of arterial aneurysms and tortuosity, mild craniofacial, skeletal and cutaneous anomalies, and early-onset osteoarthritis. AOS is caused by mutations in the SMAD3 gene. Methods A cohort of 393 patients with aneurysms without mutation in FBN1, TGFBR1 and TGFBR2 was screened for mutations in SMAD3. The patients originated from The Netherlands, Belgium, Switzerland and USA. The clinical phenotype in a total of 45 patients from eight different AOS families with eight different SMAD3 mutations is described. In all patients with a SMAD3 mutation, clinical records were reviewed and extensive genetic, cardiovascular and orthopaedic examinations were performed. Results Five novel SMAD3 mutations (one nonsense, two missense and two frame-shift mutations) were identified in five new AOS families. A follow-up description of the three families with a SMAD3 mutation previously described by the authors was included. In the majority of patients, early-onset joint abnormalities, including osteoarthritis and osteochondritis dissecans, were the initial symptom for which medical advice was sought. Cardiovascular abnormalities were present in almost 90% of patients, and involved mainly aortic aneurysms and dissections. Aneurysms and tortuosity were found in the aorta and other arteries throughout the body, including intracranial arteries. Of the patients who first presented with joint abnormalities, 20% died suddenly from aortic dissection. The presence of mild craniofacial abnormalities including hypertelorism and abnormal uvula may aid the recognition of this syndrome. Conclusion The authors provide further insight into the phenotype of AOS with SMAD3 mutations, and present recommendations for a clinical work-up.