Systematic screening of FBN1 gene unclassified missense variants for splice abnormalities.

Defects at the level of pre-mRNA splicing are a common source of genetic mutation but such mutations are not always easy to identify from DNA sequence data alone. Clinical practice has only recently begun to incorporate analysis for this type of abnormality. Some base changes at the DNA level currently viewed as unclassified variants or missense mutations may influence RNA splicing. To address this problem for fibrillin 1 (FBN1) gene missense mutations we have carried out RNA analysis and in silico analysis with splice site prediction programs on 40 cases with 36 different mutations. Direct analysis of RNA from blood was performed by cDNA preparation, PCR amplification of specific FBN1 fragments, gel electrophoresis and sequencing of the PCR products. Of the 36 missense base changes, direct RNA analysis identified 2 which caused an abnormality of splicing. In silico analysis using five splice site prediction programs did not always accurately predict the splicing seen by direct RNA analysis. In conclusion, some apparent missense mutations have an effect on splicing which can be identified by direct RNA analysis, however, in silico analysis of splice sites is not always accurate, should be carried out with more than one prediction program and results should be used with caution.

Human NDE1 splicing and mammalian brain development.

Exploring genetic and molecular differences between humans and other close species may be the key to explain the uniqueness of our brain and the selective pressures under which it evolves. Recent discoveries unveiled the involvement of Nuclear distribution factor E-homolog 1 (NDE1) in human cerebral cortical neurogenesis and suggested a role in brain evolution; however the evolutionary changes involved have not been investigated. NDE1 has a different gene structure in human and mouse resulting in the production of diverse splicing isoforms. In particular, mouse uses the terminal exon 8 T, while Human uses terminal exon 9, which is absent in rodents. Through chimeric minigenes splicing assay we investigated the unique elements regulating NDE1 terminal exon choice. We found that selection of the terminal exon is regulated in a cell dependent manner and relies on gain/loss of splicing regulatory sequences across the exons. Our results show how evolutionary changes in cis as well as trans acting signals have played a fundamental role in determining NDE1 species specific splicing isoforms supporting the notion that alternative splicing plays a central role in human genome evolution, and possibly human cognitive predominance.

Functional splicing assay shows a pathogenic intronic mutation in neurofibromatosis type 1 (NF1) due to intronic sequence exonization.

Genomic variations with no apparent effect ("neutral polymorphisms") may have a significant effect on splicing. The effect of this type of mutation is difficult to spot, unless a functional assay is undertaken. In our study, DNA sequencing of a patient with clinically defined neurofibromatosis type 1 (NF1) showed only a single polymorphism in intron 30 due to an A>G transition 279 nucleotides from the 3' splice site. Using a minigene splicing assay we conclusively show that this change produces a cryptic exon with a 3' SS defined by the nucleotide change and the unexpected activation of a very weak 5'SS. Further site directed mutagenesis studies aimed at identifying the signals involved in the cryptic exon inclusion were carried out. Interestingly we find that particular characteristics of the cryptic 5' SS are essential for its inclusion. Significantly an additional single nucleotide change disrupting the cryptic 5'ss consensus sequence rescues the effect of the pathogenetic mutation resulting in normal splicing.

Splicing in action: assessing disease causing sequence changes.

Variations in new splicing regulatory elements are difficult to identify exclusively by sequence inspection and may result in deleterious effects on precursor (pre) mRNA splicing. These mutations can result in either complete skipping of the exon, retention of the intron, or the introduction of a new splice site within an exon or intron. Sometimes mutations that do not disrupt or create a splice site activate pre-existing pseudo splice sites, consistent with the proposal that introns contain splicing inhibitory sequences. These variants can also affect the fine balance of isoforms produced by alternatively spliced exons and in consequence cause disease. Available genomic pathology data reveal that we are still partly ignorant of the basic mechanisms that underlie the pre-mRNA splicing process. The fact that human pathology can provide pointers to new modulatory elements of splicing should be exploited.

Léri-Weill syndrome associated with a pseudodicentric X;Y translocation chromosome and skewed X-inactivation: implications for genetic counselling.

A female patient of normal intelligence with short stature and Madelung deformity is reported with Léri-Weill dyschondrosteosis and a de novo pseudodicentric X;Y translocation chromosome. The phenotype is consistent with the observed deletion of the SHOX gene by FISH and molecular studies. The Y chromosome breakpoint was in the short arm but proximal to SRY, consistent with her phenotypic sex. X-inactivation studies have shown a skewed pattern in favour of the dic (X;Y) chromosome. The ARSE gene was also deleted on the dic (X;Y) chromosome but chondrodysplasia punctata was not expressed, as CDP is recessive and ARSE escapes inactivation on the normal X chromosome. Breakpoint mapping assisted in karyotype/phenotype correlation and reproductive counselling. In particular, molecular analysis showed that the putative MRX 49 gene for mental retardation is unlikely to be deleted in this case.

A case of the new overgrowth syndrome--macrocephaly with cutis marmorata, haemangioma and syndactyly.

Moore et al. [(1997) J Med Genet 70:67-73] and Clayton-Smith et al. [(1997) Clin Dysmorphol 6:291-302] have recently described a new overgrowth syndrome with macrocephaly, cutis marmorata, haemangiomas and digit syndactyly. Other features have included body asymmetry, hydrocephalus requiring shunting and developmental delay. All 22 cases reported are sporadic. We report a further case of this rare syndrome.

Novel Tandem Duplication in Exon 1 of the SNURF/SNRPN Gene in a Child with Transient Excessive Eating Behaviour and Weight Gain.

A deletion in 15q11.2 involving the SNURF/SNRPN gene is the typical finding in patients with Prader-Willi syndrome. Apart from translocations disrupting this gene, no other mutation types have been described so far. We report a patient in whom a small duplication in exon 1 of the SNURF/SNRPN gene was diagnosed which is predicted to interrupt only SNURF expression. The patient was investigated due to overgrowth, increased appetite and developmental delay in childhood. This duplication was inherited from her father who carries the duplication on his paternal chromosome 15 and also had transient excessive eating behaviour as an adolescent. RNA studies showed that the duplication introduces a premature stop codon in SNURF.

An intronic mutation in MLH1 associated with familial colon and breast cancer.

Single base substitutions can lead to missense mutations, silent mutations or intronic mutations, whose significance is uncertain. Aberrant splicing can occur due to mutations that disrupt or create canonical splice sites or splicing regulatory sequences. The assessment of their pathogenic role may be difficult, and is further complicated by the phenomenon of alternative splicing. We describe an HNPCC patient, with early-onset colorectal cancer and a strong family history of colorectal and breast tumors, who harbours a germ line MLH1 intronic variant (IVS9 c.790 +4A>T). The proband, together with 2 relatives affected by colorectal-cancer and 1 by breast cancer, have been investigated for microsatellite instability, immunohistochemical MMR protein staining, direct sequencing and Multiplex Ligation-dependent Probe Amplification. The effect of the intronic variant was analyzed both by splicing prediction software and by hybrid minigene splicing assay. In this family, we found a novel MLH1 germline intronic variant (IVS9 c.790 +4A>T) in intron 9, consisting of an A to T transversion, in position +4 of the splice donor site of MLH1. The mutation is associated with the lack of expression of the MLH1 protein and MSI in tumour tissues. Furthermore, our results suggest that this substitution leads to a complete skip of both exon 9 and 10 of the mutant allele. Our findings suggest that this intronic variant plays a pathogenic role.

Alternative splicing: good and bad effects of translationally silent substitutions.

Nucleotide variations that do not alter the protein-coding sequence have been routinely considered as neutral. In light of the developments we have seen over the last decade or so in the RNA processing and translational field, it would be proper when assessing these variants to ask if this change is neutral, good or bad. This question has been recently partly addressed by genome-wide in silico analysis but significantly fewer cases by laboratory experimental examples. Of particular relevance is the effect these mutations have on the pre-mRNA splicing pattern. In fact, alterations in this process may occur as a consequence of translationally silent mutations leading to the expression of novel splicing isoforms and/or loss of an existing one. This phenomenon can either generate new substrates for evolution or cause genetic disease when aberrant isoforms altering the essential protein function are produced. In this review we briefly describe the current understanding in the field and discuss emerging directions in the study of the splicing mechanism by integrating disease-causing splicing mutations and evolutionary changes.

An absence of cutaneous neurofibromas associated with a 3-bp inframe deletion in exon 17 of the NF1 gene (c.2970-2972 delAAT): evidence of a clinically significant NF1 genotype-phenotype correlation.

Neurofibromatosis type 1 (NF1) is characterized by cafe-au-lait spots, skinfold freckling, and cutaneous neurofibromas. No obvious relationships between small mutations (<20 bp) of the NF1 gene and a specific phenotype have previously been demonstrated, which suggests that interaction with either unlinked modifying genes and/or the normal NF1 allele may be involved in the development of the particular clinical features associated with NF1. We identified 21 unrelated probands with NF1 (14 familial and 7 sporadic cases) who were all found to have the same c.2970-2972 delAAT (p.990delM) mutation but no cutaneous neurofibromas or clinically obvious plexiform neurofibromas. Molecular analysis identified the same 3-bp inframe deletion (c.2970-2972 delAAT) in exon 17 of the NF1 gene in all affected subjects. The Delta AAT mutation is predicted to result in the loss of one of two adjacent methionines (codon 991 or 992) ( Delta Met991), in conjunction with silent ACA-->ACG change of codon 990. These two methionine residues are located in a highly conserved region of neurofibromin and are expected, therefore, to have a functional role in the protein. Our data represent results from the first study to correlate a specific small mutation of the NF1 gene to the expression of a particular clinical phenotype. The biological mechanism that relates this specific mutation to the suppression of cutaneous neurofibroma development is unknown.

A prospective study of neurofibromatosis type 1 cancer incidence in the UK.

Neurofibromatosis type 1 (NF1) is an autosomal dominant condition affecting around one in 3000 live births. The manifestations of this condition are extremely variable, even within families, and genetic counselling is consequently difficult with regard to prognosis. Individuals with NF1 are acknowledged to be at increased risk of malignancy. Several studies have previously attempted to quantify this risk, but have involved relatively small study populations. We present prospective data from 448 individuals with NF1 with a total of 5705 years of patient follow-up. These data have been collected via the UK NF1 association for patients. Demographic information on the affected individuals was cross-referenced with UK cancer registry data by the UK Office of National Statistics. The overall risk of cancer was 2.7 times higher in this cohort of NF1 patients than in the general population (95% confidence interval (CI) 1.9-3.7). The cumulative risk of a malignancy by age 50 years was 20% (95% CI 14-29%); beyond this age, the risk of cancer was not significantly elevated (P=0.27). The most frequent types of cancer were connective tissue (14% risk by age 70, 95% CI 7.8-24%) and brain tumours (7.9, 95% CI 3.9-16%). There was no statistically significant excess of cancers at other sites (P=0.22).

Linkage analysis of idiopathic generalized epilepsy (IGE) and marker loci on chromosome 6p in families of patients with juvenile myoclonic epilepsy: no evidence for an epilepsy locus in the HLA region.

Evidence for a locus (EJM1) in the HLA region of chromosome 6p predisposing to idiopathic generalized epilepsy (IGE) in the families of patients with juvenile myoclonic epilepsy (JME) has been obtained in two previous studies of separately ascertained groups of kindreds. Linkage analysis has been undertaken in a third set of 25 families including a patient with JME and at least one first-degree relative with IGE. Family members were typed for eight polymorphic loci on chromosome 6p: F13A, D6S89, D6S109, D6S105, D6S10, C4B, DQA1/A2, and TCTE1. Pairwise and multipoint linkage analysis was carried out assuming autosomal dominant and autosomal recessive inheritance and age-dependent high or low penetrance. No significant evidence in favor of linkage was obtained at any locus. Multipoint linkage analysis generated significant exclusion data (lod score < -2.0) at HLA and for a region 10-30 cM telomeric to HLA, the extent of which varied with the level of penetrance assumed. These observations indicate that genetic heterogeneity exists within this epilepsy phenotype.

Linkage analysis of idiopathic generalised epilepsy in families of probands with Juvenile Myoclonic Epilepsy and marker loci in the region of EPM 1 on chromosome 21 q: Unverricht-Lundborg disease and JME are not allelic variants.

The locus for Unverricht-Lundborg disease, EPM 1, has recently been mapped to chromosome 21q22.3. A locus, EJM 1, predisposing to idiopathic generalised epilepsy in families of probands with juvenile myoclonic epilepsy has been localised to chromosome 6p by evidence of linkage to the HLA region. However, segregation analysis suggests a two-locus model for JME and evidence has been obtained for genetic heterogeneity within the JME/IGE phenotype. EPM 1 was therefore investigated as a candidate locus in the set of families segregating for IGE and JME which do not show linkage to markers on chromosome 6p. Linkage analysis was carried out in 25 families using three microsatellite DNA markers around the EPM 1 gene region using different models of inheritance. Multipoint linkage analysis provided definite exclusion for 20cM around PFKL, the closet linked marker to EPM 1, under three out of four models tested. These results strongly suggest that the EPM 1 gene is not linked to the phenotype expressed in these families, and therefore that Unverricht-Lundborg disease and juvenile myoclonic epilepsy are not allelic variants.