Katrien Storm

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.

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.

Determination of the carrier frequency of the common GJB2 (connexin‐26) 35delG mutation in the Belgian population using an easy and reliable screening method

Mutations in the gene GJB2, encoding the gap‐junction protein connexin‐26, have been shown to be a major cause of nonsyndromic recessive deafness (NSRD). A single mutation in the GJB2 gene accounts for the majority of NSRD in many different populations. This mutation represents a deletion of a guanine within a stretch of six Gs between nucleotide positions +30 and +35 of the GJB2 cDNA (35delG). Molecular detection of the 35delG mutation is usually performed by direct sequencing analysis of PCR products, or by allele‐specific PCR analysis. To screen for this mutation, we developed an easier and more reliable method, based on the principle of PCR‐mediated site‐directed mutagenesis (PSDM), followed by a BsiYI digestion. We tested 360 unrelated unaffected Belgian individuals for heterozygosity of the 35delG mutation and found a carrier frequency of 1 in 40 (95% CI, 1 in 30 to 1 in 60). As our new screening method is simple and reliable in use, and detects a mutation responsible for a significant part of NSRD, it may find widespread use in DNA diagnostics. Hum Mutat 14:263–266, 1999.

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.

Somatic and gonadal mosaicism in Hutchinson-Gilford progeria.

We have studied a patient with Hutchinson–Gilford progeria (HGP). Sequence analysis of the LMNA gene demonstrated the presence of a c.1824 C > T (p.G608G) mutation, activating a cryptic splice donor site and leading to the formation of a truncated Lamin A protein. All molecularly characterized autosomal dominant HGP cases described so far result from de novo LMNA mutations, mostly originating on the paternal allele and are often linked with advanced paternal age. However, in our patient, the mutation was transmitted by the mother who showed somatic and germline mosaicism without HGP manifestations.

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.

Evaluation of RAD51C as cancer susceptibility gene in a large breast-ovarian cancer patient population referred for genetic testing.

Despite extensive analysis of the BRCA1 and BRCA2 genes, germline mutations are detected in <20% of families with a presumed genetic predisposition for breast and ovarian cancer. Recent literature reported RAD51C as a new breast cancer susceptibility gene. In this study, we report the analysis of 410 patients from 351 unrelated pedigrees. All were referred for genetic testing and we selected families with at least one reported case of ovarian cancer in which BRCA1&2 mutations were previously ruled out. We analyzed the coding exons, intron–exons boundaries, and UTRs of RAD51C. Our mutation analysis did not reveal any unequivocal deleterious mutation. In total 12 unique sequence variations were identified of which two were novel. Our study and others suggest a low prevalence of RAD51C mutations with an exception for some founder populations. This observation is in favor of the rare allele hypothesis in the debate over the nature of the genetic contribution to individual susceptibility to breast and ovarian cancer and further genome-wide studies in high risk families are warranted.

An interstitial deletion of chromosome 7 at band q21: A case report and review

We report on a girl with moderate developmental delay and mild dysmorphic features. Cytogenetic investigations revealed a de novo interstitial deletion at the proximal dark band on the long arm of chromosome 7 (7q21.1‐q21.3) in all analyzed G‐banded metaphases of lymphocytes and fibroblasts. Fluorescence in situ hybridization (FISH) and molecular studies defined the breakpoints at 7q21.11 and 7q21.3 on the paternal chromosome 7, with the proximal deletion breakpoint between the elastin gene (localized at 7q11.23) and D7S2517, and the distal breakpoint between D7S652 and the COL1A2 gene (localized at 7q21.3‐q22.1). Deletions of interstitial segments at the proximal long arm of chromosome 7 at q21 are relatively rare. The karyotype–phenotype correlation of these patients is reviewed and discussed. The clinical findings of patients with a deletion at 7q21 significantly overlap with those of patients with maternal uniparental disomy of chromosome 7 (matUPD(7)) and Silver–Russell syndrome (SRS, OMIM 180860). Therefore, 7q21 might be considered a candidate chromosomal region for matUPD(7) and SRS.

Myopathy and phosphorylase kinase deficiency caused by a mutation in the PHKA1 gene

Phosphorylase kinase (PhK) deficiency is the underlying cause of variable clinical symptoms depending on the tissues involved. Until today, only a few cases of myopathy associated with muscle PhK deficiency caused by a mutation in the gene encoding the alpha subunit of phosphorylase kinase (PHKA1) have been reported. We describe a male patient with myopathy and absent muscle PhK activity caused by a frameshift mutation in the gene encoding the alpha subunit of PhK on chromosome Xq12‐q13.

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.