Comparative multiplex dosage analysis in spinocerebellar ataxia type 2 patients.

We developed a new application of comparative multiplex dosage analysis (CMDA) for evaluation of the ataxin 2 gene. Expansions of the triplet CAG can cause spinocerebellar ataxia type 2 (SCA2), a neurodegenerative disease with an autosomal-dominant mode of inheritance. Molecular diagnosis of SCA2 is routinely based on the use of conventional PCR to detect the CAG expansion. However, PCR does not amplify an allele with an expansion of many triplets (>80), which is typically found in infantile and juvenile forms of SCA2, thus leading to false negatives. We propose the analysis of the ATXN2 gene by CMDA to complement existing methods currently used for the detection of large expansions of the CAG repeat. Using CMDA, the presence of any longer mutated allele in a heterozygous patient or fetus would be inferred due to dosage variation of the very frequent normal allele #22. CMDA can be completed in 1 day, at very low cost, and would be a useful tool for prenatal diagnosis and for diagnosis of presymptomatic forms of early-onset SCA2.

Multiplex ligation-dependent probe amplification detection of an unknown large deletion of the CREB-binding protein gene in a patient with Rubinstein-Taybi syndrome.

Rubinstein-Taybi syndrome is a rare autosomal dominant congenital disorder characterized by postnatal growth retardation, psychomotor developmental delay, skeletal anomalies, peculiar facial morphology, and tumorigenesis. Mutations in the gene encoding the cAMP response element-binding protein (CREB, also known as CREBBP or CBP) on chromosome 16p13.3 have been identified. In addition, some patients with low intelligence quotients and autistic features bear large deletions. Based on these observations, we used multiplex ligation-dependent probe amplification to search for large deletions affecting the CREBBP gene in a Rubinstein-Taybi syndrome patient. We identified a novel heterozygote deletion removing five exons (exons 17-21), encoding the histone acetyltransferase domain. We propose the use of multiplex ligation-dependent probe amplification as a fast, accurate and cheap test for detecting large deletions in the CREBBP gene in the sub-group of Rubinstein-Taybi syndrome patients with low intelligence quotients and autistic features.

Analysis of the gastrin-releasing peptide receptor gene in Italian patients with autism spectrum disorders.

The gastrin-releasing peptide receptor (GRPR) was implicated for the first time in the pathogenesis of Autism spectrum disorders (ASD) by Ishikawa-Brush et al. [Ishikawa-Brush et al. (1997): Hum Mol Genet 6: 1241-1250]. Since this original observation, only one association study [Marui et al. (2004): Brain Dev 26: 5-7] has further investigated, though unsuccessfully, the involvement of the GRPR gene in ASD. With the aim of contributing further information to this topic we have sequenced the entire coding region and the intron/exon junctions of the GRPR gene in 149 Italian autistic patients. The results of this study led to the identification of four novel point mutations, two of which, that is, C6S and L181F, involve amino acid changes identified in two patients with ASD and Rett syndrome, respectively. Both the leucine at position 181 and the cysteine at position 6 are strongly conserved in vertebrates. C6S and L181F mutant proteins were expressed in COS-7 and BALB/3T3 cells, but they did not affect either GRP's binding affinity or its potency for stimulating phospholipase C-mediated production of inositol 1,4,5-trisphosphate. In summary, our results do not provide support for a major role of the GRPR gene in ASD in the population of patients we have studied. However, there is a potential role of C6S and L181F mutations on GRPR function, and possibly in the pathogenesis of the autistic disorders in the two patients.

Preliminary studies on the molecular basis of hyperphenylalaninemia in Egypt.

Mutation analysis at the phenylalanine hydroxylase (PAH) locus was undertaken in 56 Egyptian hyperphenylalaninemic patients. Selected screening for 11 known mutations and denaturing Gradient gel electrophoresis (DGGE) analysis of the entire coding sequence and exon/intron boundaries led to the identification of a new mutation (I224T), four previously described mutations, and several polymorphisms. Overall, 18 mutant alleles could thus be characterized. In contrast to the high mutation detection rate typical of the DGGE-based scanning approach, only 6 of 16 mutant alleles tested were identified. Since BH4 deficiency could not be excluded in any of these patients, the latter results may be explained by the occurrence of mutations affecting the genes controlling the synthesis and recycling of tetrahydrobiopterin: the cofactor of PAH. An alternative hypothesis is also discussed.

MtDNA control region and RFLP data for Sicily and France.

The forensic application of mtDNA typing requires large databases which are regionally well defined. To further this aim, we have typed mtDNA in a sample of 111 French and 106 Sicilians. The French were typed for both hypervariable segments (HVR1 and HVR2) of the mtDNA control region, whereas the Sicilians were only typed for HVR1, but in addition for the coding region RFLP markers for mtDNA groups H, I, J, K, L, M, T, U, V and X. In both samples, the predominant sequence type by far was the Cambridge reference sequence. Comparing HVR1 sequences, we found that the French sample was twice as diverse as the Sicilian sample as measured by sequence matches. A further set of sequence match comparisons including the French, Sicilian, and the published British mtDNA samples, demonstrate that sequence matching probabilities within samples differ by less than a factor of 2 from the matching probabilities between samples.