Lieve Vits

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

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.

Genotype-phenotype correlation in L1 associated diseases.

The neural cell adhesion molecule L1 (L1CAM) plays a key role during embryonic development of the nervous system and is involved in memory and learning. Mutations in the L1 gene are responsible for four X linked neurological conditions: X linked hydrocephalus (HSAS), MASA syndrome, complicated spastic paraplegia type 1 (SP-1), and X linked agenesis of the corpus callosum. As the clinical picture of these four L1 associated diseases shows considerable overlap and is characterised by Corpus callosum hypoplasia, mental Retardation, Adducted thumbs, Spastic paraplegia, and Hydrocephalus, these conditions have recently been lumped together into the CRASH syndrome. We investigate here whether a genotype-phenotype correlation exists in CRASH syndrome since its clinical spectrum is highly variable and numerous L1 mutations have been described. We found that (1) mutations in the extracellular part of L1 leading to truncation or absence of L1 cause a severe phenotype, (2) mutations in the cytoplasmic domain of L1 give rise to a milder phenotype than extracellular mutations, and (3) extracellular missense mutations affecting amino acids situated on the surface of a domain cause a milder phenotype than those affecting amino acids buried in the core of the domain.

The clinical spectrum of mutations in L1, a neuronal cell adhesion molecule

Mutations in the gene encoding the neuronal cell adhesion molecule L1 are responsible for several syndromes with clinical overlap, including X-linked hydrocephalus (XLH, HSAS), MASA (mental retardation, aphasias, shuffling gait, adducted thumbs) syndrome, complicated X-linked spastic paraplegia (SP 1), X-linked mental retardation-clasped thumb (MR-CT) syndrome, and some forms of X-linked agenesis of the corpus callosum (ACC). We review 34 L1 mutations in patients with these phenotypes.

ASSIGNMENT OF X-LINKED HYDROCEPHALUS TO XQ28 BY LINKAGE ANALYSIS

X-linked recessive hydrocephalus (HSAS) occurs at a frequency of approximately 1 per 30,000 male births and consists of hydrocephalus, stenosis of the aqueduct of Sylvius, mental retardation, spastic paraparesis, and clasped thumbs. Prenatal diagnosis of affected males by ultrasonographic detection of hydrocephalus is unreliable because hydrocephalus may be absent antenatally. Furthermore, carrier detection in females is not possible because they are asymptomatic. Using four families segregating HSAS, we performed linkage analysis with a panel of X-linked probes that detect restriction fragment length polymorphisms. We report here that HSAS, in all tested families, is closely linked to marker loci mapping in Xq28 (DXS52, lod = 6.52 at theta of 0.03; F8, lod = 4.32 at theta of 0.00; DXS15, lod = 3.40 at theta of 0.00). These data assign HSAS to the gene-dense chromosomal band Xq28 and allow for both prenatal diagnosis and carrier detection by linkage analysis.

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 optimized DHPLC protocol for molecular testing of the EXT1 and EXT2 genes in hereditary multiple osteochondromas

Hereditary multiple osteochondromas (MO) is an autosomal dominant bone disorder characterized by the presence of bony outgrowths (osteochondromas or exostoses) on the long bones. MO is caused by mutations in the EXT1 or EXT2 genes, which encode glycosyltransferases implicated in heparan sulfate biosynthesis. Standard mutation analysis performed by sequencing analysis of all coding exons of the EXT1 and EXT2 genes reveals a mutation in approximately 80% of the MO patients. We have now optimized and validated a denaturing high‐performance liquid chromatography (DHPLC)‐based protocol for screening of all EXT1‐ and EXT2‐coding exons in a set of 49 MO patients with an EXT1 or EXT2 mutation. Under the optimized DHPLC conditions, all mutations were detected. These include 20 previously described mutations and 29 new mutations – 20 new EXT1 and nine new EXT2 mutations. The protocol described here, therefore, provides a sensitive and cost‐sparing alternative for direct sequencing analysis of the MO‐causing genes.

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.

Mapping of the gene for X-linked liver glycogenosis due to phosphorylase kinase deficiency to human chromosome region Xp22

X-linked liver glycogenosis (XLG) is a glycogenosis due to deficient activity of phosphorylase kinase (PHK) in liver. PHK consists of four different subunits, alpha, beta, gamma, and delta. Although it is unknown whether liver and muscle PHK subunits are encoded by the same genes, the muscle alpha subunit (PHKA) gene was a likely candidate gene for the mutation responsible for this X-linked liver glycogenosis as it was assigned to the X chromosome at q12-q13. Linkage analysis with X-chromosomal polymorphic DNA markers was performed in two families segregating XLG. First, multipoint linkage analysis excluded the muscle PHKA region as the site of the XLG mutation. Second, evidence was obtained for linkage between the XLG locus and DXS197, DXS43, DXS16, and DXS9 with two-point peak lod scores Zmax = 6.64, 3.75, 1.30, and 0.88, all at theta max = 0.00, respectively. Multipoint linkage results and analysis of recombinational events indicated that the mutation responsible for XLG is located in Xp22 between DXS143 and DXS41.

Frequency of the phenylalanine deletion (ΔF508) in the CF gene of Belgian cystic fibrosis patients

Cloning and sequencing of the CF gene has identified a three‐base‐pair deletion (ΔF508) responsible for CF in the majority of CF patients (Kerem et al. 1989). We have used the polymerase chain reaction with oligonucleotide primers bridging the ΔF308 deletion to analyze the presence or absence of this mutation in the Belgian CF population. The ΔF306 mutation was present in 80% (57 on 71) of CF chromosomes from 36 unrelated Belgian CF families from the region of Antwerp. This mutation was associated with haplotype B for the KM.19–XV‐2c RFLPs as 93% (53 on 57) of the CF chromosomes with the Δ306 mutation carried haplotype B.