MASA syndrome is due to mutations in the neural cell adhesion gene L1CAM.

MASA syndrome is a recessive X-linked disorder characterized by mental retardation, adducted thumbs, shuffling gait, aphasia and, in some cases, hydrocephalus. Since it has been shown that X-linked hydrocephalus can be caused by mutations in L1CAM, a neuronal cell adhesion molecule, we performed an L1CAM mutation analysis in eight unrelated patients with MASA syndrome. Three different L1CAM mutations were identified: a deletion removing part of the open reading frame and two point mutations resulting in amino acid substitutions. L1CAM, therefore, harbours mutations leading to either MASA syndrome or HSAS, and might be frequently implicated in X-linked mental retardation with or without hydrocephalus.

The full mutation in the FMR-1 gene of male fragile X patients is absent in their sperm.

Fragile X syndrome is characterized at the molecular level by amplification of a (CGG)n repeat and hypermethylation of a CpG island preceeding the open reading frame of the fragile X gene (FMR-1) located in Xq27.3. Anticipation in this syndrome is associated with progressive amplification of the (CGG)n repeat from a premutation to a full mutation through consecutive generations. Remarkably, expansion of the premutation to the full mutation is strictly maternal. To clarify this parental influence we studied FMR-1 in sperm of four male fragile X patients. This showed that only the premutation was present in their sperm, although they had a full mutation in peripheral lymphocytes. This might suggest that expansion of the premutation to the full mutation in FMR-1 does not occur in meiosis but in a postzygotic stage.

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 lle367Asn substitution. This de novo mutation is absent in the patient's 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.

Restoring the phenotype of fragile X syndrome: insight from the mouse model.

A mouse model for the fragile X syndrome, the most common form of inherited mental retardation, was generated a number of years ago. It shows characteristics compatible with the clinical symptoms of human patients. These include pathological changes such as macroorchidism, behavioral problems, and diminished visuo-spatial abilities. To investigate whether the fragile X syndrome is a potentially correctable disorder, several groups attempted to 'rescue' the knockout mutation by introduction of an intact copy of the FMR1 gene in the knockout mouse. Two different types of rescue mice have been created by injection of constructs based on FMR1 cDNA or on FMR1 genomic DNA. Several pathological, behavioral and cognitive function tests were performed on these two different rescue mouse lines to compare their characteristics with those of the knockout and control littermates. Each rescue line resembled the control in some aspects though neither of the 2 lines was a full 'rescue', e.g. resemble the control in all aspects investigated. Thus, rescue of some aspects of the phenotype has been achieved by introduction of FMR1 constructs in the fragile X knockout mice. The results implicate that, even if FMR1 production is cell type specific, the quantity of the FMRP expression is highly critical as overproduction may have a harmful effect.

Variable regions of chromosome 11 loss in different pathological tissues of a patient with the multiple endocrine neoplasia type I syndrome.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant inherited disorder characterized by nodular proliferation of the parathyroid glands and tumors of the anterior pituitary gland, the endocrine pancreas, and the neuroendocrine cell system of the gut. Loss of the putative tumor suppressor effect of the MEN1 gene is probably responsible for the development of MEN1-associated tumors. We report here a genetic study of a female MEN1 patient with the association of nodular hyperplasia of two parathyroid glands, an insulinoma, multiple duodenal gastrinomas, a prolactinoma, and a gastric carcinoid. We performed loss of heterozygosity (LOH) studies of chromosome 11 on all affected tissues except the insulinoma. Allelic losses of chromosome 11 were detected in several tumors, but the chromosomal regions of LOH were different, suggesting that different somatic mutational events are involved in the pathogenesis of these tumors. LOH of chromosome 11 was also detected in the prolactinoma of this patient, which indicates that the MEN1 gene has a tumor suppressor effect in the pituitary.

Neuroanatomy of the fragile X knockout mouse brain studied using in vivo high resolution magnetic resonance imaging.

Magnetic resonance imaging (MRI) of the brain of fragile X patients, the most frequent form of inherited mental retardation, has revealed abnormalities in the size of specific brain structures, including the cerebellar vermis, the hippocampus, and the ventricular system. We intended to quantify the differences observed in the patient studies in the fragile X knockout mouse model, which is a good model for the disease, paralleling the human disorder in having cognitive deficits, macro-orchidism, and immature dendritic spines. Therefore we set up MRI of the mouse brain which allowed us to measure the size of the brain structures reported to be abnormal in human fragile X patients in the mouse model. We did not find evidence for size alterations in various brain regions of the fragile X mouse model, but the method described may find a wide application in the study of mutant mouse models with neurological involvement.

Mildly impaired water maze performance in male Fmr1 knockout mice.

Fmr1 knockout mice constitute a putative model of fragile X syndrome, the most common form of heritable mental disability in humans. We have compared the performance of transgenic mice with an Fmr1 knockout with that of normal littermates in hidden- and visible-platform water maze learning, and showed that knockouts exhibit subnormal spatial learning abilities and marginal motor performance deficits. During 12 training trials of the hidden-platform task, escape latency and path length decreased significantly in knockouts and control littermates, and no effect of genotype was found. During four ensuing reversal trials, however, significant differences were found between knockouts and control littermates both in escape latency and path length. During the visible-platform condition, the reversal trials also revealed a difference between knockouts and normal littermates in escape latency, but not in path length. Possibly due to marginal motor incapacity, knockouts swam significantly slower than controls during these latter trials. During both probe trials of the hidden-platform task, knockouts as well as normal littermates spent more time in the target quadrant than in the other quadrants, and percent of time spent in the target quadrant was the same in both groups; swimming velocity was not significantly different between knockouts and normal littermates during these trials. Entries in the target area during the probe trials did show a significant effect of genotype on number of entries. The present results largely confirm and extend our previous findings. Impaired spatial abilities in Fmr1 knockouts might have been due to relatively low response flexibility or high memory interference in Fmr1 knockouts. It remains unclear, however, which brain region or neurochemical system might be involved in these disabilities. We conclude that Fmr1 knockout mice might be a valid model of fragile X mental retardation.

Severe mental retardation and macroorchidism without mutation in the FMR1 gene.

Only one missense mutation, an Ile304Asn, has been reported in the fragile X gene (FMR1). This mutation is located in the second KH domain of FMR1, and has led to the discovery of the function of the FMR1 gene product as an RNA-binding protein. The patient carrying this mutation has profound mental retardation, macroorchidism, and an "acromegalic" face with prominent supraorbital ridges, enlarged jaw, heavy brow ridges, thick lips, and a broad nose. We have studied the possible involvement of FMR1 in two maternal half-brothers with a phenotype similar to that of the patient with the Ile304Asn mutation. Both brothers had an identical number of CGG repeats in the normal size-range, and shared the same maternal Xq27 haplotype. Southern blot analysis with two overlapping FMR1 cDNA clones, spanning the total FMR1 open reading frame, showed no major deletions, insertions, or gross rearrangements. Single-strand conformation pattern (SSCP) analysis of the KH domains showed no aberrant patterns. The total open reading frame of the FMR1 gene was cloned and sequenced, but no mutation was found. Northern blot analysis showed mRNA in the normal size-range, and immunocytochemistry on individual lymphocytes indicated that FMRP, the protein product of FMR1, was present. In conclusion, it is unlikely that FMR1 plays a role in the phenotype of this patient. Other genes may be responsible for the combination of mental retardation and macroorchidism.

CAG repeat contraction in the androgen receptor gene in three brothers with mental retardation.

We report on three brothers with mental retardation and a contracted CAG repeat in the androgen receptor (AR) gene. It is known that expansion of the CAG repeat in this gene leads to spinal and bulbar muscular atrophy (SBMA or Kennedy disease); however, contracted repeats have not yet been implicated in disease. As the range of the length of CAG repeats in the AR gene, like those of other genes associated with dynamic mutations, follows a normal distribution, the theoretical possibility of disease at both ends of the distribution should be considered.

Postmortem examination of two fragile X brothers with an FMR1 full mutation.

Large expansions of the CGG repeat in the 5' untranslated region of the FMR1 gene are found in patients with the fragile X syndrome. Amplified CGG repeats in FMR1 are unstable and show intergenerational increase from mother to offspring. The exact timing of repeat amplification, however, is unknown. We have compared the extent of CGG expansion in various tissues of this deceased fragile X patient, and found only limited variation in repeat expansion. The repeat was fully methylated in all tissues examined. Therefore, no evidence for extensive mitotic expansion of the CGG repeat during fetal or postnatal life of a fragile X patient was found, in contrast to dynamic mutations caused by CAG/CTG repeat expansion. Extensive pathological examination of this patient and his affected brother revealed no evidence for specific abnormalities relevant to fragile X syndrome; cerebellar hypoplasia, which has been reported in this disorder, was not evident in either patient.

Long-term potentiation in the hippocampus of fragile X knockout mice.

To gain more insight in the physiological function of the fragile X gene (FMR1) and the mechanisms leading to fragile X syndrome, the Fmr1 gene has been inactivated in mice by gene targeting techniques. In the Morris water maze test, the Fmr1 knockout mice learn to find the hidden platform nearly as well as the control animals, but show impaired performance after the position of the platform has been modified. As malperformance in the Morris water maze test has been associated with impaired long-term potentiation (LTP), electrophysiological studies were performed in hippocampal slices of Fmr1 knockout mice to check for the presence of LTP. Judged by field extracellular excitatory postsynaptic potential recordings in the CA1 hippocampal area, Fmr1 knockout mice express LTP to a similar extent as their wild type littermates during the first 1-2 hr after high frequency stimulation. Also, short-term potentiation (STP) was similar in both types of mice. To investigate whether Fmr1 is involved in the latter stages of LTP as an immediate early gene, we compared Fmr1 mRNA quantities on northern blots after chemical induction of seizures. A transient increase in the transcription of immediate early genes is thought to be essential for the maintenance of LTP. As no increase in Fmr1 mRNA could be detected, neither in cortex nor in total brain, during the first 2 1/2 hr after pentylenetetrazol-induced seizures, it is unlikely that Fmr1 is an immediate early gene in mice. In conclusion, we found no evidence for a function of FMR1 in STP or LTP.

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.

Spatial learning, contextual fear conditioning and conditioned emotional response in Fmr1 knockout mice.

Fmr1 knockout mice are an animal model for fragile X syndrome, the most common form of heritable mental retardation in humans. Fmr1 knockout mice exhibit macro-orchidism and cognitive and behavioural deficits reminiscent of the human phenotype. In the present study additional behavioural and cognitive testing was performed. Knockouts and control littermates were subjected to a spatial learning test using a plus-shaped water maze. Animals had to learn the position of a hidden escape platform during training trials. The position of this platform was changed during subsequent reversal trials. Previously reported deficits in reversal learning were replicated, but we also observed significant differences during the acquisition trials. A plus-shaped water maze experiment with daily changing platform positions failed to provide clear evidence for a working memory impairment, putatively underlying the spatial learning deficits. Two different test settings were used to examine the reported deficit of Fmr1 knockout mice in fear conditioning. Conditioned fear responses were observed in a contextual fear test, and the ability to acquire an emotional response was tested by means of response suppression in a conditioned emotional response procedure. Neither protocol revealed significant differences between controls and knockouts.

Distal myopathy with upper limb predominance caused by filamin C haploinsufficiency.

OBJECTIVE: In this study, we investigated the detailed clinical findings and underlying genetic defect in 3 presumably related Bulgarian families displaying dominantly transmitted adult onset distal myopathy with upper limb predominance. METHODS: We performed neurologic, electrophysiologic, radiologic, and histopathologic analyses of 13 patients and 13 at-risk but asymptomatic individuals from 3 generations. Genome-wide parametric linkage analysis was followed by bidirectional sequencing of the filamin C (FLNC) gene. We characterized the identified nonsense mutation at cDNA and protein level. RESULTS: Based on clinical findings, no known myopathy subtype was implicated in our distal myopathy patients. Light microscopic analysis of affected muscle tissue showed no specific hallmarks; however, the electron microscopy revealed changes compatible with myofibrillar myopathy. Linkage studies delineated a 9.76 Mb region on chromosome 7q22.1-q35 containing filamin C (FLNC), a gene previously associated with myofibrillar myopathy. Mutation analysis revealed a novel c.5160delC frameshift deletion in all patients of the 3 families. The mutation results in a premature stop codon (p.Phe1720LeufsX63) that triggers nonsense-mediated mRNA decay. FLNC transcript levels were reduced in muscle and lymphoblast cells from affected subjects and partial loss of FLNC in muscle tissue was confirmed by protein analysis. CONCLUSIONS: The FLNC mutation that we identified is distinct in terms of the associated phenotype, muscle morphology, and underlying molecular mechanism, thus extending the currently recognized clinical and genetic spectrum of filaminopathies. We conclude that filamin C is a dosage-sensitive gene and that FLNC haploinsufficiency can cause a specific type of myopathy in humans.

Multiplex ligation-dependent probe amplification to detect subtelomeric rearrangements in routine diagnostics.

Subtelomeric rearrangements are believed to be responsible for 5-7% of idiopathic mental retardation cases. Due to the relative complexity and high cost of the screening methods used till now, only preselected patient populations including mostly the more severely affected cases have been screened. Recently, multiplex ligation-dependent probe amplification (MLPA) has been adapted for use in subtelomeric screening, and we have incorporated this technique into routine diagnostics of our laboratory. Since the evaluation of MLPA as a screening method, we tested 275 unselected patients with idiopathic mental retardation and detected 12 possible subtelomeric aberrations: a der(11)t(11;20)(qter;qter), a 19pter duplication, a der(18)t(18;10)(qter; pter), a 15qter deletion, a 8pter deletion, a 6qter deletion, a der(X)t(X;1)(pter;qter), a der(X)t(X;3)(pter;pter), a 5qter duplication, a 3pter deletion, and two 3qter duplications. The patients can be subdivided into two groups: the first containing de novo rearrangements that are likely related to the clinical presentation of the patient and the second including aberrations also present in one of the parents that may or may not be causative of the mental retardation. In our patient cohort, five (1.8%) subtelomeric rearrangements were de novo, three (1.1%) rearrangements were familial and suggestively disease causing, and four (1.5%) were possible polymorphisms. This high frequency of subtelomeric abnormalities detected in an unselected population warrants further investigation about the feasibility of routine screening for subtelomeric aberrations in mentally retarded patients.

Apparent regression of the CGG repeat in FMR1 to an allele of normal size.

The fragile X syndrome is the result of amplification of a CGG trinucleotide repeat in the FMR1 gene and anticipation in this disease is caused by an intergenerational expansion of this repeat. Although regression of a CGG repeat in the premutation range is not uncommon, regression from a full premutation (> 200 repeats) or premutation range (50-200 repeats) to a repeat of normal size (< 50 repeats) has not yet been documented. We present here a family in which the number of repeats apparently regressed from approximately 110 in the mother to 44 in her daughter. Although the CGG repeat of the daughter is in the normal range, she is a carrier of the fragile X mutation based upon the segregation pattern of Xq27 markers flanking FMR1. It is unclear, however, whether this allele of 44 repeats will be stably transmitted, as the daughter has as yet no progeny. Nevertheless, the size range between normal alleles and premutation alleles overlap, a factor that complicates genetic counseling.

An EcoRI RFLP in the 5' region of the human NF1 gene.

Von Recklinghausen neurofibromatosis or type 1 neurofibromatosis (NF1), is one of the most common autosomal dominant disorders. NF1 is characterized by neurofibromas, café-au-lait spots and Lisch nodules of the iris. The NF1 gene is located in 17q11.2. The restriction fragment length polymorphism reported here will be useful in linkage analysis in NF1 families.

Gene conversion between red and defective green opsin gene in blue cone monochromacy.

Blue cone monochromacy is an X-linked condition in which the function of both the red pigment gene (RCP) and the green pigment gene (GCP) is impaired. Blue cone monochromacy can be due to a red/green gene array rearrangement existing of a single red/green hybrid gene and an inactivating C203R point mutation in GCP. We describe here a family with blue cone monochromacy due to the presence of the C203R mutation in both RCP and GCP. The flanking sequences of the C203R mutation in exon 4 of RCP were characteristic for GCP, indicating that this mutation was transferred from GCP into RCP by gene conversion.

Incomplete EcoRI digestion may lead to false diagnosis of fragile X syndrome.

Molecular diagnosis of fragile X syndrome is usually performed using Southern blot analysis of DNA digested with EcoRI. In the course of diagnostic studies, we observed that a specific EcoRI restriction site in the fragile X gene (FMR1) is sometimes refractory to digestion, generating additional fragments on a Southern blot suggestive of a full mutation in FMR1. This may lead to a false-positive diagnosis of fragile X syndrome. Such additional bands are avoided by the use of HindIII instead of EcoRI. Therefore, we recommend the use of HindIII for the molecular diagnosis of fragile X syndrome.

Segregation of the fragile X mutation from an affected male to his normal daughter.

We report here a family in which the fragile X mutation segregates from an affected grandfather through his normal daughter to an affected grandson. The grandson shows clinical and cytogenetic expression of fragile X syndrome due to a full mutation (large methylated insertion) in the fragile X gene (FMR-1). The mother shows a premutation (small unmethylated insertion) in her FMR-1 gene as the sole manifestation of the fragile X syndrome. The grandfather expresses the fragile X syndrome at the clinical and cytogenetic level, whereas he is mosaic for a methylated full mutation and an unmethylated premutation. The absence of expression of the fragile X mutation when transmitted through an expressing male might present further evidence for genomic imprinting of the FMR-1 gene. Alternatively, it is possible that the grandfather transmitted his premutation to his daughter due to germline mosaicism with both the premutation and the full mutation present in his sperm.