Objective: Myotonic dystrophy type 1 and 2 (DM1/DM2) are multisystemic diseases with common cognitive deficits beside the cardinal muscular symptoms. We performed a comprehensive analysis of cerebral abnormalities to compare the neuropsychological defects with findings in different imaging methods in the same cohort of patients. Methods: Neuropsychological investigations, structural cerebral MRI including brain parenchymal fraction (BPF) and voxel-based morphometry (VBM), and 18F-deoxy-glucose PET (FDG-PET) were performed in patients (20 DM1 and 9 DM2) and matched healthy controls, and analyzed using statistical parametric mapping (SPM2). Results: DM1 and DM2 patients showed typical neuropsychological deficits with a pronounced impairment of nonverbal episodic memory. Both patient groups showed a reduction of the global gray matter (measured by BPF), which could be localized to the frontal and parietal lobes by VBM. Interestingly, VBM revealed a bilateral hippocampal volume reduction that was correlated specifically to both a clinical score and episodic memory deficits. VBM also revealed a pronounced change of thalamic gray matter. White matter lesions were found in >50% of patients and their extent was correlated to psychomotor speed. FDG-PET revealed a frontotemporal hypometabolism, independent of the decrease in cortical gray matter. All abnormalities were similar in both patient groups but more pronounced for DM1. Conclusions: Our results suggest that 1) some of the characteristic cognitive deficits of these patients are linked to specific structural cerebral changes, 2) decreases in gray matter and metabolism are independent processes, and 3) the widespread brain abnormalities are more pronounced in DM1.

Myotonic dystrophy type 1 and 2 (DM1/DM2) are slowly progressive multisystemic diseases. Core features are myotonia, muscle weakness and atrophy, with a more benign course in DM2. Further clinical findings are cardiac arrhythmia, cataracts, endocrine changes and cognitive deficits.

Gene mapping data indicate that the human X chromosome is enriched in genes that affect both, higher cognitive efficiency and reproductive success. This raises the question whether these functions are ancient, or whether conserved X-linked genes were recruited to new functions. We have studied three X-linked mental retardation (XLMR) genes by RNA in situ hybridization in mouse and in chicken, in which these genes are autosomal: Rho guanine nucleotide exchange factor 6 (ARHGEF6), oligophrenin (OPHN1), and p21 activated kinase 3 (PAK3). In the mouse these genes are specifically expressed in telencephalic regions. Their orthologues in the chicken gave patterns of similar specificity in ancient parts of the brain, i.e. cerebellum and mesencephalon, but were not expressed in the telencephalon. Also in the testes, specific expression was only found in mouse, not in chicken. These data are interpreted such that certain genes on the X chromosome gained novel functions during evolution.

Thirteen new cases of a pericentric inversion 2 collected from different laboratories are reported. In addition 41 cases of a pericentric inversion 2 were reviewed from the literature. The pooled data were analysed using Weinberg's proband method to evaluate the risk of a carrier for either children with congenital anomalies or reproductive wastage. In the "corrected" sample of 166 lifeborn offspring of carriers of a pericentric inversion 2 there were five who showed phenotypic anomalies and two died a few hours after delivery. The reported anomalies are heterogeneous and probably reflect the basic risk of any couple for abnormal lifeborn offspring. There has been no observation of a lifeborn who inherited an unbalanced recombination of a parental pericentric inversion 2. A carrier of a pericentric inversion 2 obviously has an increased risk for reproductive wastage. This is indicated by (1) an increase of the rate of spontaneous abortions and (2) an increase of the rate of index patients ascertained because of previous miscarriages. The risk of a carrier of a pericentric inversion 2 for a spontaneous abortion or a stillbirth may be about twice the basic risk of the general population.

The CGG repeat in the 5' untranslated region of the fragile X mental retardation 1 gene (FMR1) exhibits remarkable instability upon transmission from mothers with premutation alleles. A collaboration of 13 laboratories in eight countries was established to examine four issues concerning FMR1 CGG-repeat instability among females with premutation (approximately 55-200 repeats) and intermediate (approximately 46-60 repeats) alleles. Our central findings were as follows: (1) The smallest premutation alleles that expanded to a full mutation (>200 repeats) in one generation contained 59 repeats; sequence analysis of the 59-repeat alleles from these two females revealed no AGG interruptions within the FMR1 CGG repeat. (2) When we corrected for ascertainment and recalculated the risks of expansion to a full mutation, we found that the risks for premutation alleles with <100 repeats were lower than those previously published. (3) When we examined the possible influence of sex of offspring on transmission of a full mutation-by analysis of 567 prenatal fragile X studies of 448 mothers with premutation and full-mutation alleles-we found no significant differences in the proportion of full-mutation alleles in male or female fetuses. (4) When we examined 136 transmissions of intermediate alleles from 92 mothers with no family history of fragile X, we found that, in contrast to the instability observed in families with fragile X, most (99/136 [72.8%]) transmissions of intermediate alleles were stable. The unstable transmissions (37/136 [27.2%]) in these families included both expansions and contractions in repeat size. The instability increased with the larger intermediate alleles (19% for 49-54 repeats, 30.9% for 55-59, and 80% for 60-65 repeats). These studies should allow improved risk assessments for genetic counseling of women with premutation or intermediate-size alleles.

Myotonic dystrophy (DM1) and proximal myotonic myopathy (PROMM or DM2) are two distinct muscular disorders with multisystemic involvement. Both have previously been reported to be associated with cognitive impairment and white matter lesions detected by cerebral magnetic resonance imaging (MRI). In this study, the extent of brain atrophy was investigated in vivo in ten DM1 and nine PROMM patients in comparison to age-matched healthy controls for each group. The diagnosis was confirmed by DNA analysis of all patients. As a quantitative marker, the ratio of brain parenchymal to intracranial volume, called brain parenchymal fraction (BPF), was calculated from 3-dimensional MRI data using an automated analysis technique. Compared to age-matched healthy controls (mean BPF 0.852 +/- 0.032), the BPF in DM1 patients (0.713 +/- 0.031) was highly significantly decreased (P < 0.001). In contrast, the PROMM patients (mean BPF 0.792 +/- 0.029) showed only slightly decreased BPF values (P < 0.05). BPF was not significantly correlated to any of the clinical or genetic parameters in both diseases (disease duration, motor score, educational level, and number of CTG repeats in the expanded allele). In summary, global brain atrophy was demonstrated to occur in both diseases, but was more severely manifestated in DM1 patients.

The major causes of fragile X syndrome are mutational expansion of the CGG repeat in the FMR1 gene, hypermethylation, and transcriptional silencing. Most fragile X embryos develop somatic mosaicism of disease-causing "full" expansions of different lengths. Homogeneity of the mosaic patterns among multiple tissues in the same individual indicates that these previously unstable expansions acquire mitotic stability early in fetal life. Since mitotic stability is found strictly associated with hypermethylation in adult tissues, current theory has fixed the time of instability to developmental stages when fully expanded CGG repeats exist in an unmethylated state. We used murine embryocarcinoma (EC) cells (PC13) as a model system of pluripotent embryonic cells. Hypermethylated and unmethylated full expansions on human fragile X chromosomes were transferred from murine A9 hybrids into EC cells, by means of microcell fusion. As demonstrated in the present study for the first time, even full expansion alleles that were fully methylated and stable in the donors' fibroblasts and in A9 became demethylated, reactivated, and destabilized in undifferentiated EC hybrids. When destabilized expansions were reintroduced from EC cells into A9, instability was reversed to stability. Our results strongly support the idea that fully expanded alleles are initially unstable and unmethylated in the human embryo and gain stability upon genetic or epigenetic change of the embryonic cells.

The ninth in this series of international workshops was held at “Le Bischenberg”, Obernai, Strasbourg, France, on 23–25 August, 1999, the same exquisite location of the fifth workshop organized by Jean-Louis Mandel with the exciting reports on the FMR1-gene discovery. The meeting was organized by the European XLMR Consortium (Jamel Chelly (Paris), Jean-Pierre Fryns (Leuven), Ben Hamel (Nijmegen), Claude Moraine (Tours), Hans-Hilger Ropers (Berlin), and Jean-Louis Mandel (Illkirch). Local organizers were Claude Stoll (Strasbourg) and Jean-Pierre Fryns (Leuven)), although major part of the work of organization was ably performed by Marleen Van Leemputten. The meeting was well attended, with 133 participants from 24 countries. The meeting opened with an invited lecture by Gillian Turner on “The History of X-Linked Mental Retardation.” The first part of the meeting was on X-linked mental retardation (XLMR) (36 platform presentations) with two clinical sessions chaired by Jean-Pierre Fryns, Lisbeth Tranebjaerg, Charles Schwartz and Didier Lacombe, a session on XLMR-mapping chaired by Laurent Villard and John Mulley, a session on new and candidate genes chaired by Hans-Hilger Ropers and Ben Hamel, a session on XLMR-molecular screening chaired by Jean-Louis Mandel and Claude Moraine, and a session on XLMR-new technologies (microarrays in gene finding and mutation analysis) chaired by Ted Brown and Hans Van Bolhoven with excellent contributions by Jean-Louis Mandel, Hans-Hilger Ropers and David Nelson. Ben Hamel presented the Update 2000 on syndromic XLMR genes [Hamel et al., 2000]. The XLMR part ended by a session on “Cellular Molecular Mechanisms in Neurodevelopment and -Function” chaired by Jamel Chelly and David Nelson, with two invited lectures by Ger Ramakers [Ramakers, 2000] and Ligun Luo, and the “Update 2000 on XLMR Linkage Data and New Genes” by Jamel Chelly [Chelly, 2000]. The Fragile X part of the conference (29 platform presentation) included a clinical session (chair: Gillian Turner and Michael Partington), a psychology session (chair: Martine Borghgraef and Claude Stoll), a session on population screening (chair: Angela Vianna-Morgante and James MacPherson), and two sessions on the molecular basis of Fragile X syndrome (chair: Ben Oostra, Peter Steinbach, André Hanauer, Paul Hagerman). The session on 37 posters, on XLMR and Fragile X syndrome, was chaired by Francis Rousseau and Gene Fisch. At the end of the meeting there was a lively discussion to determine the site of the next meeting, that will be in Italy at the Lago di Garda and will be hosted by Daniela Toniolo and Giovanni Neri. The session contained 6 presentations. Maarit Peippo described a family with variable severity of the same symptoms in a mother and her son manifesting microcephaly, hearing loss, epilepsy, ataxia and cerebellar hypoplasia. No genetic mapping-information was given. Muscle-biopsy and VEP were normal. The question was raised whether it could be X-linked or autosomal dominant inheritance. Based on the experience from a later presentation during the workshop, by Elke Holinski-Feder, the possibility of submicroscopic chromosomal telomeric deletion is worth exploring. Didier Lacombe reported 3 cases of the so called W Syndrome. He discussed the degree of clinical variation, that could lead to lack of complete ascertainment from previous estimates of the occurrence of the syndrome. Another two presentations by, respectively, Roger Stevenson, and Charles Schwartz, both from the Greenwood Genetic Center group, tried to analyze 2 clinical findings in greater detail: the hypotonic face and spasticity. Even if the hypotonic face is a nonspecific finding it is possible to analyze the upper, mid and lower third of the face of each patient in a more systematic fashion, but no scoring system was suggested. Based on this systematic approach, families with new syndromes had been picked as candidates for mutations in the ATRX-gene, that definitely has a hypotonic face as a very prominent finding. In the upper part of the face, hypotonia manifests as puffiness, perhaps ptosis of eyelids, or a thick upper eyelid. In the mid-part of the face it manifests as a thick helix and thick alae nasi. In the lower part of the face it manifests as an open mouth and prominent lips. Examples were given of syndromes where this is very prominent such as ATRX, Chudley-Lowry and Coffin-Lowry, Juberg-Marsidi, Carpenter, Holmes-Gang, Finemann-Smith-Myers syndromes, whereas it is not present in X-linked lissencephaly. Sabine Dassay discussed the clinical characteristics in her families with FG syndrome. Previously genetic heterogeneity along the X chromosome had been indicated, but she discussed whether the actual family suffered from a different syndrome, based on clinical dissimilarity. She had performed X inactivation studies in 10 families, and in 5 of them found random X inactivation. Finally, Connie Schrander-Stumpel reported a family with complicated spastic paraplegia and evidence for the disease not mapping to the L1-CAM region, but rather pointing to a possible locus on Xp21.1–Xq21.3 and Xq22.1–Xq26.3. Candidate genes, such as the PLP gene and Synapsin 1 gene, were investigated and no mutations were found. The responsible gene remained to be identified. The initial two presentations in this session dealt with clinical delineation of the mitochondria problems associated with the Mohr-Tranebjaerg syndrome (MTS). Three subsequent talks presented data on two new XLMR syndromes and the analysis of a family with ATR16. The final presentation was an update of the XLMR syndromes. MTS comprises deafness, dystonia and MR. The gene for MTS had previously been cloned [Jin et al., 1996] and was named DDP for deafness/dystonia peptide. Recently, Koehler et al. [1999] provided evidence that DDP, also known as TIM8p, mediates the import of transporters from the cytoplasm into the mitochondrial inner membrane, showing MTS to be a mitochondrial disorder. In view of these findings, two groups presented their analysis, clinically and at the mitochondrial level, of several patients with MTS. Ben Hamel (Nijmegen, Holland) described 2 families with 3 males affected with MTS. In one family, a missense mutation (C233G) in DDP gave rise to deafness, mild MR and dystonia. Results of muscle biopsy and metabolic screening were normal, however, as were MRI and ophthalmologic studies. The second family had a young male with deafness and mild MR associated with severe behavior problems. His uncle was also deaf, had eye movement problems, decreased vision after age 30, cerebellar ataxia, and hyperreflexia without dystonia or MR. This family had a deletion that removed exon 2 of DDP as well as other genomic material beyond DDP. A muscle biopsy revealed an increased number of fat vacuoles that might be associated with decreased mitochondrial ATP production. Metabolic tests demonstrated elevated alanine and results by an oral glucose tolerance test were normal. This family is of some interest due to the variable presentation in the nephew and uncle not all of which could be explained by the progressive nature of the disease in the uncle because the latter did not have MR. Lisbeth Tranebjaerg presented the clinical and laboratory findings in a set of 8 patients with MTS. Seven of the patients had truncating mutations of DDP whereas the eighth, also studied by Hamel's group, had a missense mutation (C233G) that disrupts the ZnF motif in the gene. Of interest was the lack of any apparent genotype/phenotype correlation among these patients. Only one of four patients studied exhibited ragged red fibers on muscle biopsy whereas 4/4 had increased numbers and subsarcolemmal aggregates of mitochondria. Furthermore, there was only a slight reduction in ATP production in the muscle without any complex specificity. One other interesting finding was noted: the absence of TIM13p, that is related to TIM8p/DDP, in the MTS patients analyzed. The significance of this is not understood at present. Elke Holinski-Feder presented a 5 generation family in which the affected individuals had moderate MR and minor anomalies. Linkage analysis showed this family to actually link to 16p13.3 rather than the X-chromosome. Subsequent analysis demonstrated a subtelomeric deletion of 16p in the affected and a balanced translocation t(3q;16p) in the carriers. Thus, the condition in this family fits into the ATR16 syndrome (MR, mild ∝-thalassemia, and non-specific minor anomalies). No mention was made of any family members having trisomy 3q. The fourth paper was to have been presented by David Cabezas, but difficulties with US Immigration authorities prevented him from attending the meeting. The abstract dealt with the localization between DXS424(Xq23) and DXS1047 (Xq25) of a new XLMR syndrome delineated by the presence of short stature, small testes, muscle wasting and tremor. Frank Kooy described the linkage analysis in an XLMR family in which the affected males had exhibited mild MR, choreoathetosis, episodic and spastic hypertonia and arachnodactyly. This new XLMR syndrome mapped to Xp11, flanked by DXS1201 (Xp11.3) and DXS1190 (Xp11.2). Suggested potential candidate genes included GPR34, a G-protein receptor and KCND1, an ion channel gene. The last presentation of the session was an XLMR syndrome update by Ben Hamel. At this time (August 23, 1999), 124 specific XLMR were identified. His summation of new XLMR syndromes, refined localizations, and isolation of new genes is presented elsewhere in this report [Hamel et al., 2000]. Since the localizations of MRX1 and MRX2 in 1988, the number of mapped but overlapping loci for nonspecific X-linked mental retardation (MRX) has increased to 75 worldwide (August 1999). These are genetically heterogeneous families. They segregate genes for cognitive impairment in men (and women where inactivation is skewed). There are no syndromal characteristics. A two-point or multipoint lod score of at least +2 is mandatory to demonstrate linkage (preferably with exclusion from the remainder of the X chromosome). Localizations are the intervals between the closest flanking recombination points in each family. These large MRX families remain an extremely rare but invaluable resource for mutation screening leading to the identification of genes for familial MRX. The present indications are that large numbers of MRX genes await discovery and that most will account for very few of the families that map across their locations. This means that true MRX genes from among the candidate genes tested may remain unidentified. This can be due to chance, where families with that defective gene are absent from the sample of families tested. Testing may be required using significantly more than 75 MRX families to exclude a candidate gene as an MRX gene. Smaller families large enough to infer sex linkage are now valued for the purpose of identification of genes responsible for familial MRX. This is especially true when supported by suggestive linkage to, and exclusion from, specific regions of the X chromosome. The families ascertained so far have been skewed heavily toward the severely or moderately retarded range of cognitive dysfunction. FMR2 is the only gene identified to date that is consistently responsible for milder mental impairment. It is also responsible for a relatively large proportion of the families that have nonspecific XLMR. Ascertainment of such families would have been difficult without the FRAXE marker given the relatively mild and variable presence of measurable mental impairment. The location of other genes for similar mild dysfunction of cognitive ability will remain elusive due to the difficulties in both the ascertainment of these families and in making the distinction between affected and normal men. Locating autosomal genes for nonspecific mental retardation remains as a future challenge, but is an approachable goal using homozygosity mapping in consanguineous families with affected sibships. John Mulley reported on a woman with a balanced translocation t(X;12)(q24;q15) and bipolar affective disorder, seizures and MR. A gene interrupted by the translocation breakpoint had been identified: glutamate receptor subunit 3 (GR1A3) that is a neurotransmitter and involved in neuronal plasticity. No mutations, however, have been found so far in one family with an X-linked bipolar affective disorder nor in MRX27. Additional families with non-specific XLMR will be analyzed. The Heidelberg group presented results of the study of 14 Xp22.3 deletion patients (4 with MR, 10 without MR). A gene VCX-A (Variably Charged, X chromosome) was isolated, belonging to a gene family that contains at least 4 paralogs on Xp22.3: VCX-A, B, B1, and C. All deletion-patients without MR had either VCX-A or B or both, whereas those with MR lacked both VCX-A and B, suggesting that one intact copy is enough for normal intelligence. In 5 linked families, however, no mutations were found. Helger Yntema reported on Xq21 deletions, associated with a contiguous gene syndrome consisting of deafness (DFN3, gene POU3F4), choroideremia (CHM, gene REP-1) and MRX. A novel gene was isolated, that was called RSK4, based on its homology to the known RSKs (e.g., RSK2 in Coffin-Lowry syndrome and MRX). No mutations have been found so far in a large cohort of familial MR, compatible with X-linked inheritance. Lin Jun presented a male patient with a syndromic form of MR and a pericentric inversion inv(X)(p21.1;q22). Molecular analysis of the Xq breakpoint (Xp breakpoint contained no MR gene) demonstrated a novel gene: TLP1, homologous to TAP gene, a nuclear transport gene. The function of TLP1 is unknown. It is expressed in fetal brain. Results of mutation screening are pending. Vincent Des Portes performed a systematic mutational analysis of Doublecortin in 11 females (10 sporadic and 1 familial) with subcortical laminar heterotopia (SCLH). Doublecortin is the gene involved in X-linked SCLH/lissencephaly syndrome. In 10/11 cases mutations (nonsense, missense, splice site) were found. A geno/phenotype correlation was not found. Doublecortin mutations were also found in 3/16 males with sporadic lissencephaly. As late breaking news, Jamel Chelly presented exciting data on 2 new genes involved in non-specific XLMR. The first gene named IL1 receptor accessory protein-like = IL1RAPL (because of its homology to IL1RACP), was isolated studying two overlapping microdeletions in Xp22.1–p21.3 in patients with non-specific XLMR. A stopmutation was found in only one (out of 150 tested probands) small family with non-specific XLMR. The function of IL1RAPL is still largely unknown, but it might be involved in neurotransmitter release and in the formation of long-term potentiation in the hippocampus and synaptic plasticity. The second gene named Tetraspanin (belonging to the transmembrane 4 superfamily = TM4SF), was detected after cloning the X breakpoint in a female with MR and autistic behavior and a balanced t(X;2)(p11.4;p21.3). Subsequently, in 2 families with non-specific XLMR mutations (missense and nonsense respectively) were found. A major characteristic of the Tetraspanin family is its ability to associate with other surface molecules such as α and β integrins. Its function, however, is also largely unknown, but Tetraspanin could play a role in actin cytoskeleton organization and activation of MAP kinase cascades. X-linked mental retardation (XLMR) is a very heterogeneous condition and comprises specific entities (MRXS) and non-specific mental retardation (MRX). More than 70 clinically different entities have been described in the MRXS subgroup and 20 of the corresponding genes are cloned. MRXS, however, seems to be much less frequent than MRX that accounts for more than 2/3 of all XLMRs. Collaborative linkage studies performed in MRX families have shown more than 15 linkage intervals that may correspond to MRX genes, suggesting great genetic heterogeneity. Up to now, molecular studies performed by 1) FISH physical mapping and positional cloning on X-chromosome rearrangements in mentally retarded individuals, and 2) by testing candidate genes that map in the X-chromosome regions defined by linkage studies, have identified only 7 genes mutated in MRX families. At this moment, a great proportion of the different X-chromosome intervals that were delimited for MRX genes by linkage studies are still not known to contain candidate genes or to correspond to one or more of the X-chromosomal rearrangement breakpoints. Systematic testing of the 7 identified MRX genes has shown mutations in a low percentage of families: as presented by Judith Mallolas for PaK3 and by Jamel Chelly's group for oligophrenin 1. These data suggest that the high frequency of MRX in the different populations studied may result from: 1) some frequent particular mutation(s) in one or more known or still unknown MRX gene(s); 2) non-frequent mutations in numerous, still unknown MRX genes, some of them possibly not involved in X-chromosomal rearrangements; or 3) particular mutations in genes that are known to be involved in other X-linked pathologies and especially in MRX. Allelism seems to be a common phenomenon in X-linked pathologies, and at least three examples have been reported so far: L1CAM mutated in X-linked hydrocephalus, MASA and SPG2; ATP7A mutated in Menkes and occipital horn syndromes; PLP mutated both in SPG1 and in Pelizaeus-Merzbacher syndrome. On the other hand, intrafamilial clinical variability is frequently observed in MRX families with known or unknown genes. Therefore, it is suggested to test known MRXS genes in families with specific XLMR conditions mapping in the same X-chromosomal region, but clinically different from the initially described syndrome e.g., 1) Laurent Villard reported in this session a mutation in the XNP gene, different in localization and nature from that found in the ATRX syndrome, in a family originally published as Smith-Fineman-Myers syndrome, or 2) Sylvie Jacquot described a particular mutation in the RSK2 gene in a family with non-specific mental retardation, without obvious Coffin-Lowry syndrome findings. For the same reason, Hans van Bokhoven suggested studying MRX families by searching mutations in some parts of MRXS genes that are not specifically mutated in the syndromal form. Finally, it can not be excluded that the frequency of mutations observed in the different genes involved in MRX may be different in different populations. Collaborative studies as reported by Gena Nothwang from the European XLMR consortium will greatly enhance both 1) the number of X-chromosomal rearrangements available for searching new genes disrupted or inactivated at their breakpoint, and 2) the number of mapped MRX families available to test the corresponding candidate genes or others for different mutations at different sites. Currently data suggest that there may be up to 100 genes involved in nonspecific XLMR (MRX) and perhaps an equal number underlying syndromal mental retardation. The known genes each have a modest contribution to the prevalence of MRX (0.5–1%), that confers major problems to the implementation/inclusion of these genes in a DNA-diagnostic routine. With today's DNA-diagnostic protocols it is feasible and sensible to test only those genes that are more frequently causally involved in mental retardation, such as the FMR1 gene. Consequently, only a small fraction of all mental retardation may be explained molecularly. To increase the number of positive DNA-based diagnoses it will be necessary to develop novel high-throughput mutation analysis protocols, suitable for the identification of unknown mutations in large numbers of genes. Several novel developments that are potentially useful for this were discussed in this session. These new techniques may be useful not only for mutation analysis of known MRX genes, but may be applied also for the identification of additional MRX genes. Microarray technology has a high potential for fulfilling that demand. Several methods are used to produce microarrays of either oligonucleotides or cDNA fragments. Affymetrix Gene Chip technology is well suited for screening major genes that are involved in frequently occurring diseases, e.g., p53 in tumors, but obviously this does not apply to MRX. To date, no microarrays are available that were designed for analysis of MRX gene(s). Until such arrays become available alternative high-throughput approaches have to be used, of which D-HPLC and direct sequencing on 96 capillary machines seem to be the most robust ones. People in the MRX field, however, are not sitting back waiting for new technology to come by. For example, H.H. Ropers and J.L. Mandel both alluded to their initiatives to develop cDNA microarrays for the detection of new mutations in known and yet unknown MRX genes. According to Ropers, such arrays may be used for linkage studies (by using SNPs), deletion screening (by comparative genomic hybridization, CGH), mutation screening and expression profiling. J.L. Mandel also envisages that expression profiling with such arrays may be used for the identification of additional MRX genes. The idea behind this is that many of the genes that will be involved in normal brain development will be expressed in cultured cells as in EBV-transformed lymphoblasts and fibroblasts. A mutation in an MRX gene may cause a significant reduction (e.g., by nonsense-mediated RNA decay) or even the total absence (e.g., due to a microdeletion) of transcription. In the experiment, fluorescent cDNA probes will be prepared from total RNA isolated from these cell lines and hybridized onto cDNA microarrays in which for example all X-chromosomal genes or a selection of brain-specific genes are represented. Qualitative and quantitative determination of the hybridization pattern will thus demonstrate a cell-specific expression profile. By comparing the expression profiles of cells derived from MRX patients with those of control individuals it may be possible to identify genes that are mutated in patients or genes that are active in cellular processes involving an MRX gene. Although little experience is yet available, it seems that the sensitivity of this technique has yet to be improved to be successful. Microarrays are being considered also for mutation detection. H.H. Ropers is working along two lines. The first is a primer-elongation method, in which gene-specific oligonucleotides containing an NH2 group at their 5′-terminus are attached to coated glass slides. For each target sequence wild-type and mutant oligonucleotides are designed that contain at their 3′ end the normal or changed nucleotide, respectively. The oligonucleotides can be elongated by one nucleotide (primer extension) in a reaction that contains denatured PCR products of the desired target gene as a template. This method has been applied successfully in a blind experiment in which patients were screened for a variety of all known mitochondrial mutations. Again, this method seems to be suitable only for mutation detection in genes that are frequently involved in human disease. The second protocol that was presented may have a more general application: the construction of a universal mutation detection chip. This chip will consist of all 4096 possible 6-mer oligonucleotides. To increase the stability of homoduplex formation these oligonucleotides will contain a peptide backbone, so-called PNAs. Fluorescent PCR products from exons of one or more genes are hybridized to the PNA-based microarray and analysis of the hybridization pattern should reveal possible mutations. D. Nelson discussed a third high-throughput mutation detection protocol. Rather than being based on high-density arrays, this method uses microspheres to covalently link desired target molecules. The microspheres, a product of Luminex (Austin, TX), are color-coded by the use of two dyes in different combinations, that currently produces 100 different color-coded microspheres. Similar as in the array-based method, primers can be attached onto these microspheres and elongated by one (fluorescent) nucleotide. The combination of a unique microspheres and the fluorescent signal produced by the addition of one of the four possible nucleotides yields a new unique fluorescent signal that can be recognized by laser technology. The reactions are carried in liquid environment with all possible microspheres and DNA targets to be analyzed in one tube. After the reactions, individual particles are separated by flow-cytometry and individually analyzed by laser (20,000 microspheres per second). Because of the relative ease of linking oligonucleotides to the microspheres this method seems to be an attractive alternative for microarray-based methods. The use of more dyes will exponentially increase the number of unique color-coded microspheres, and thus the potential for their use in the mutation analysis of genetically complex disorders such as XLMR. Of course, other considerations such as speed, reliability, reproducibility, sensitivity and costs are crucially important aspects to consider which method will be most attractive for implementation in a regular DNA diagnostic routine. Over the last two years, the repertoire of genes involved in non-specific XLMR has expanded very rapidly. At first sight and because of the molecular heterogeneity of primary defects, it would be difficult to point out common physiopathological mechanisms underlying cognitive impairment and intellectual handicap that are the main manifestations in mental retardation conditions. Despite this high degree of apparent heterogeneity, however, some MRX gene products, such as RhoGAP (oligophrenin1) [Billuart et al., 1998], PAK3 [Allen et al., 1998], and TM4SF2/integrin complex [Zemni et al., 2000], seem to have specific biological functions that regulate shared as well as subgroup-specific signaling pathways involving Rho family GTPases to modulate actin cytoskeleton organization (Fig. 1). This potential common characteristic may suggest that the cellular primary defect associated with loss of function mutations in some MRX genes could be the impaired ability of actin cytoskeleton to drive neurite outgrowth leading therefore to aberrant neuronal morphogenesis, dendritogenesis and connectivity between neuronal cells. This hypothesis is supported by reliable data showing that axonal extension, dendritogenesis and synaptogenesis are driven by actin polymerization within the growth cone, a highly dynamic structure at the tip of the axones and dendrites, consisting of filipodial and lamellipodial protrusions that respond to both positive and negative external guidance cues [Luo et al., 1996; Zipkin et al., 1997; Threadgill et al., 1997; Hall et al., 1998]. The identification of further MRX genes is essential for the better comprehension of the physiopathological mechanisms underlying MR. Putative signal transduction pathways in growth cones and schematic illustration of potential interconnections between MRX genes through their involvement in the regulation of Rho family GTPases activity and actin cytoskeleton organization. Extracellular guidance cues (L) interact with growth cone receptors (R), that in turn activate signaling cascades involving Rho-like GTPases. Activated, GTP-bound, RhoGTpases stimulate filopodia and lamellipodia formation or induce growth cone collapse. Dysfunction of PAK3, RhoGAP, TM4SF2 and FGD1 proteins (bold and underlined proteins) is associated with mental retardation. FGD1, the product of the faciogenital dysplasia or Aarskog-Scott syndrome locus [Pasteris et al., 1994], is a guanine nucleotide exchange factor (GEF) [Zheng et al., 1996]. About 30% of patients affected by this syndrome have nonspecific mental retardation [Fryns., 1992]. N-WASP [Miki et al., 1998], neural protein highly homologous to Wiskott-Aldrich syndrome protein (WASP) [Derry et al., 1994]. The tetraspanin/integrin complexes can activate Rac and Rho GTPases through mechanisms reviewed by Gioncotti [1997]. Dotted arrows indicate oversimplified pathways for which proteins and biochemical partners involved are not fully identified. This session started with two papers with a very similar theme. The first was by Christine de Die-Smulders and her colleagues in Maastricht and Leuven and the second was by Claude Stoll and Yves Alembik from Strasbourg. Between them these groups presented 27 children (24 boys and 3 girls) with the fragile X syndrome where the diagnosis had been missed by the referring clinician. There was no single explanation. In some the diagnosis had just not been considered sometimes because the clinical manifestations were not specific, in some because no family history had been taken, or if taken and found positive, the implications had been disregarded and in some the diagnosis was just incorrect (e.g., the Sotos syndrome, Ehlers-Danlos syndrome, the Prader-Willi syndrome). The presenters of both papers made strong pleas for continued education on the fragile X syndrome to all professionals in medicine, psychology and education with exhortations to have a high index of suspicion about the fragile X syndrome and if in doubt to test appropriately for it. The third paper was by Rebecca O'Connor and her colleagues in Denver, Colorado. These workers studied 12 boys out of 108 with the fragile X syndrome chosen because they had severe language delay defined as failure to use meaningful three word phrases by 5 years of age. No significant differences were found in these 12 compared to the rest of the group in physical or behavioral index scores and CTG repeat number, the presence of partial or full methylation or the presence of less than 15% expression of FMR1 protein in peripheral lymphocytes. It was noted that none of the boys with substantial amounts of FMRP had severe language delay. Elizabeth Hi

Using methylation-sensitive restriction enzymes, we characterized the methylation pattern on the 5' side of the CTG repeat in the DMPK gene of normal individuals and of patients affected with myotonic dystrophy, showing expansions of the repetitive sequence. The gene segment analyzed corresponds to the genomic SacI-HindIII fragment carrying exons 11-15. There is constitutive methylation in intron 12 at restriction sites of SacII and HhaI, localized 1,159-1,232 bp upstream of the CTG repeat, whereas most, if not all, of the other sites of SacII, HhaI, and HpaII in this region are unmethylated, in normal individuals and most of the patients. In a number of young and severely affected patients, however, complete methylation of these restriction sites was found in the mutated allele. In most of these patients, the onset of the disease was congenital. Preliminary in vivo footprinting data gave evidence for protein-DNA contact in normal genes at an Sp1 consensus binding site upstream of the CTG repeat and for a significant reduction of this interaction in cells with a hypermethylated DMPK gene.

We report on further cases of high functioning fragile X males showing decreased expression of FMR1 protein, absence of detectable methylation at the EagI site in the FMR1 gene promoter, and highly unusual patterns of fragile X mutations defined as smear of expansions extending from premutation to full mutation range. Very diffuse and therefore not easily detectable patterns of full mutations were also observed on prenatal testing using DNA from chorionic villi sampled at a time of development when full mutations were still unmethylated in this particular tissue. In the search for possible determinants of such unusual patterns, repeat expansions in the premutation and in the lower full mutation range were identified on genomic PstI blots previously prepared for fragile X DNA testing. Cases with 130 or more triplets, and a number of shorter repeats, were reinvestigated on EcoRI plus EagI digests. Among the 119 expansions, there were 22 in our sample showing either blurred bands or smears on PstI blots. This particular characteristic was strongly associated with the coincidence of a repeat size of more than 130 triplets and absence of EagI site methylation. Our data set also includes cases of mosaic patterns consisting of smears of unmethylated expansions to more than 130 CGGs and of clear bands of methylated expansions. We therefore suggest that in fragile X syndrome unusual smeared patterns of mutations result from somatic instability of larger repeats under circumstantial absence of repeat methylation.

American Journal of Medical GeneticsVolume 64, Issue 2 p. 266-267 Letter to the Editor DNA methylation and triplet repeat stability: New proposals addressing actual questions on the CGG repeat of fragile X syndrome Doris Wöhrle, Doris Wöhrle Department of Medical Genetics University of Ulm, Ulm, GermanySearch for more papers by this authorSabine Schwemmle, Sabine Schwemmle Department of Medical Genetics University of Ulm, Ulm, GermanySearch for more papers by this authorPeter Steinbach, Corresponding Author Peter Steinbach Department of Medical Genetics University of Ulm, Ulm, GermanyDepartment of Medical Genetics, Parkstr. 11, 89073 Ulm, GermanySearch for more papers by this author Doris Wöhrle, Doris Wöhrle Department of Medical Genetics University of Ulm, Ulm, GermanySearch for more papers by this authorSabine Schwemmle, Sabine Schwemmle Department of Medical Genetics University of Ulm, Ulm, GermanySearch for more papers by this authorPeter Steinbach, Corresponding Author Peter Steinbach Department of Medical Genetics University of Ulm, Ulm, GermanyDepartment of Medical Genetics, Parkstr. 11, 89073 Ulm, GermanySearch for more papers by this author First published: 9 August 1996 https://doi.org/10.1002/ajmg.1320640202Citations: 12AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume64, Issue29 August 1996Pages 266-267 RelatedInformation

We demonstrate here that somatic variation of CGG repeat length is based on a mosaic of cells with different but stable FMR-1 alleles and does not reflect permanent mitotic instability. The length of a particular allele in an individual cell was maintained in progeny cells establishing a clone. The mutation patterns of multiple repeats in the DNA of fetal tissues were identical and did not significantly change during proliferation in vitro. It is proposed that genotype mosaicism and expansion to full mutation are generated post-conceptionally by the same molecular mechanism in a particular window of early development.

Molecular genetic analysis of the transmission of mutations in 73 families with fragile X (one of the largest samples evaluated so far) has confirmed previous hypotheses that the fragile X syndrome results from two consecutive mutational steps, designated ''premutation'' and ''full fragile X mutation''. These mutations give rise to expansions of restriction fragments, most probably by amplification of the FMR-1 CGG repeat. Premutations are identified by small expansions that apparently have no effect on either the clinical or the cellular phenotype. Full mutations are reflected by large expansions and hypermethylation of the expanded gene region. All males showing large expansions were affected. Individuals with full mutations also expressed the fragile X, with only one exception. An affected ''mosaic'' male, showing a predominance of premutated fragments in his leukocytes, was shown to be fragile-X-negative on different occasions. About 50% of heterozygotes with full mutations were reported by clinicians to be mentally retarded. Conversion of the premutation to the full mutation may occur at oogenesis, as previously suggested, or after formation of a zygote at an early transitional stage in development when the CGG repeat behaves as a mitotically unstable element on maternally derived/imprinted X chromosomes carrying a premutation of sufficient repeat length.

To elucidate the function of the FMR1 gene, we applied RNA in situ hybridization to cryosections of mice from different developmental stages. The murine Fmr-1 was found transcribed in a ubiquitous manner with an expression pattern similar to glyceraldehyd phosphate dehydrogenase, Gapdh, which was used as a control gene. A significant difference in the Fmr-1 expression pattern, however, was markedly enhanced expression specifically confined to the testis and the fetal ovary. In the immature and mature testis an elevated level of Fmr-1 expression is found in type A1 spermatogonia. Expression in the testis is observed in fetal life, reaches the highest level in the immature testis, and declines early in adult life. In the mature ovary no specific Fmr-1 expression signal was found but enhanced levels were seen in the fetal ovary. At this developmental stage proliferation of oogonia takes place. It is suggested that FMR1 serves a special function during germ cell proliferation in males and females. These findings are discussed in the light of the current observation that fragile X patients produce only sperm with a premutation sized allele. Two hypotheses are put forward: (1) In males lack of FMR1 function results in a premeiotic defect preventing spermatogonia with a full mutation to reach meiosis. A fragile X mutation can be passed on to offsprings only as a premutation (selection hypothesis). (2) Transition of a premutation allele to full mutation occurs in a postzygotic stage after separation of the germ line and is restricted to soma cells (restriction hypothesis). Expression of FMR1 in proliferating germ cells is in line with both hypothesis.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene of 110 cystic fibrosis (CF) patients from the south-west of Germany was screened for 12 different mutations. This analysis resulted in an identification of 79% of all CF mutations and a complete genotype in 66% of the families. The most common mutation found was delta F508 (67%). Another 5 mutations accounted for a further 12.5% (4% G542X; 3% R553X; 3% N1303K; 2% 1717-1 G-->A; 0.5% G551D) whereas 6 mutations (R117H, A455E, delta I507, S549I, S549N, and R1162X) were not found. Fifty-four (49%) patients were delta F508 homozygotes and 18 (16.5%) were compound heterozygotes for delta F508 and one of the rarer mutations. These frequencies differ slightly from those found in the north of Germany and considerably from those reported from the south of Europe, which seems to be consistent with a north to south decline of the relative abundance of delta F508. Two patients, age 6 and 25 years, were compound heterozygotes for G542X and N1303K. The clinical features of the 6 year old were characterised by severe gastrointestinal and as yet only mild pulmonary complications whereas the 25 year old manifested severe pulmonary and gastrointestinal symptoms indicating that the N1303K mutation of the C-terminal CFTR nucleotide binding fold significantly impairs protein function in both the pancreas and the lungs.

A gene designated "FMR-1" has been isolated at the fragile-X locus. One exon of this gene is carried on a 5.1-kb EcoRI fragment that exhibits length variation in fragile-X patients because of amplification of or insertion into a CGG-repeat sequence. This repeat probably represents the fragile site. The EcoRI fragment also includes an HTF island that is hypermethylated in fragile-X patients showing absence of FMR-1 mRNA. In this paper, we present further evidence that the FMR-1 gene is involved in the clinical manifestation of the fragile-X syndrome and also in the expression of the cellular phenotype. A deletion including the HTF island and exons of the FMR-1 gene was detected in a fragile X-negative mentally retarded male who presented the clinical phenotype of the fragile-X syndrome. The deletion involves less than 250 kb of genomic DNA, including DXS548 and at least five exons of the FMR-1 gene. These data support the hypothesis that loss of function of the FMR-1 gene leads to the clinical phenotype of the fragile-X syndrome. In the fragile-X syndrome, there are pathogenetic mechanisms other than amplification of the CGG repeat that do have the same phenotypic consequences.

We report on 12 prenatal diagnoses performed between weeks 10 and 13 on normal women with a well-documented family history of the Martin-Bell syndrome. Seven were obligate and three were potential carriers. One male and 2 female fetuses were found to be fragile X [fra(X)]-positive. The diagnoses were confirmed in fibroblasts or lymphocytes after interruption or postnatally. In one fra(X)-negative female fetus, the analysis of linked DNA markers indicated that most probably she was a heterozygote. Reexamination after birth gave a fra(X)-positive result. Hence this was a case of a false-negative prenatal fra(X) result. The occurrence of false-negative cytogenetic results represents a common problem that limits the sensitivity of prenatal diagnostics in the Martin-Bell syndrome. A study of linked DNA markers can improve the reliability of negative cytogenetic results in first trimester prenatal diagnosis. In case of doubt, the chromosomes could be reexamined after fetal blood sampling.

The fragile X syndrome is the most common cause of familial mental retardation and is characterized by a fragile site at the end of the long arm of the X chromosome. The unusual genetics and cytogenetics of this X-linked condition make genetic counseling difficult. DNA studies were of limited value in genetic counseling, because the nearest polymorphic DNA loci had recombination fractions of 12% or more with the fragile X mutation, FRAXA. Five polymorphic loci have recently been described in this region of the X chromosome. The positions of these loci in relation to FRAXA were defined in a genetic linkage study of 112 affected families. The five loci--DXS369, DXS297, DXS296, IDS, and DXS304--had recombination fractions of 4% or less with FRAXA. The closest locus, DXS296, was distal to FRAXA and had a recombination fraction of 2%. The polymorphisms at these loci can be detected in DNA enzymatically digested with a limited number of restriction endonucleases. A strategy for DNA studies which is based on three restriction endonucleases and on five probes will detect one or more of these polymorphisms in 94% of women. This strategy greatly increases the utility of DNA studies in providing genetic advice to families with the fragile X syndrome.