SMN1 gene copy number analyses for SMA healthy carriers in Italian population.

The routine molecular test for spinal muscular atrophy (SMA) diagnosis is based on the detection of a homozygous deletion of exons 7 and 8 of the telomeric copy of the survival motor neuron gene (SMN1). The presence of the centromeric copy of the SMN gene (SMN2) does not allow the detection of the hemizygous absence of the SMN1 gene, which characterizes the disease carriers. The demand for a quantitative SMN1 test is permanently growing because there is a high incidence of carriers. The disease is severe and to date there are no effective pharmacological treatments. Here, we present a non-radioactive assay based on real time quantitative polymerase chain reaction. We analyzed eight SMA patients, 14 SMA relatives and 50 health individuals from Southern Italy by real time quantitative method in order to identify haploid deletion occurring in SMA carriers. SMN1 copy number was determined by the comparative threshold cycle method (ΔΔCt). The results confirmed the deletion in all homozygous patients and permitted an evaluation of the number of alleles in the healthy carriers. This method is fast, reproducible, and enables us to discriminate carriers from healthy homozygous, which is impossible with normal techniques.

Putative role of specific JAG1 gene exons in modulating clinical features in patients with leukoencephalopathy.

The aim of this study was to investigate the possible role of JAG1 gene mutations in modulating clinical features in patients with CADASIL-like phenotype which resulted negative for NOTCH3 gene mutations. Sixty-six CADASIL-like patients without NOTCH3 gene mutations were investigated for 5 out of 26 exons of the JAG1 gene, whose mutations were implicated in central nervous system vascular abnormalities. PCR was performed with primers specific for exons 3, 4, 13, 23 and 24 comprising the intron-exon boundaries. Amplicons were then analyzed by denaturing high performance liquid chromatography (DHPLC). The exons showing a variant DHPLC profile were directly sequenced. The sequence of exons 3, 4 and 23 revealed the presence of four already described polymorphisms in JAG1. 1001C/T (g.16015 C>T) in exon 4 was found in 9 patients, IVS23+18delT (g.33147 delT) in 29 patients, IVS3-15T/C (g.15852 T>C) in 17 patients, IVS2-43C/T (g.10532 C>T) in 1 patient; both the polymorphism 1001C/T and IVS3-15T/C were found in 3 patients. No mutations were found. These data demonstrate absence of correlation between mutations in specific JAG1 gene exons and clinical features in patients with CADASIL-like phenotype.

A novel mutation in the CLN1 gene in a patient with juvenile neuronal ceroid lipofuscinosis.

We describe the clinical, neuropathological and molecular findings from a patient affected with neuronal ceroid lipofuscinosis with a juvenile onset (JNCL). She was a 9-year-old right-handed girl with a normal birth and early developmental milestones. At the age of 4 the early symptoms began. Skin biopsy showed granular osmiophilic deposits (GRODs). Because JNCL with GRODs is caused by mutations in the CNL1 gene, we performed a molecular investigation by direct sequencing of nine exons of the CNL1 gene. This analysis revealed a novel mutation in homozygous form in the exon 7 that caused an aminoacid substitution at codon 222 (Leu --> Pro). Direct sequencing of the exon 7 in both parents showed the same substitution in heterozygous form.