A.L. Gabriele

A novel Angiogenin gene mutation in a sporadic patient with amyotrophic lateral sclerosis from southern Italy

Mutations in the Angiogenin gene (ANG) linked to 14q11.2 have been recently discovered to be associated with Amyotrophic Lateral Sclerosis (ALS) in Irish and Scottish populations. In our study we investigated the role of ANG gene in ALS patients from southern Italy. We found a novel mutation in the signal peptide of the ANG gene in a sporadic patient with ALS (SALS). The molecular analysis of the ANG gene also demonstrated an allelic association with the rs11701 single nucleotide polymorphism (SNP) in familial ALS (FALS) but not in SALS patients. Our finding supports the evidence that the ANG gene is involved in ALS.

CADASIL: Extended polymorphisms and mutational analysis of the NOTCH3 gene

CADASIL is a cerebrovascular disease caused by mutations in the NOTCH3 gene. Most mutations result in a gain or loss of cysteine residue in one of the 34 epidermal growth factor‐like repeats in the extracellular domain of the Notch3 protein, thus sparing the number of cysteine residues. To date, more than 130 different mutations in the NOTCH3 gene have been reported in CADASIL patients, of which 95% are missense point mutations. Many polymorphisms have also been identified in the NOTCH3 coding sequence, some of them leading to amino acid substitutions. The aim of the present study was to analyze the NOTCH3 gene in a large group of patients affected by leukoencephalopathy and to investigate the presence of genetic variants. The molecular analysis revealed several nucleotide alterations. In particular, we identified 20 different mutations, 22 polymorphisms, and 8 genetic variants of unknown pathological significance never reported previously. We hope that this NOTCH3 gene mutational analysis, performed in such a significant number of unrelated and related patients affected by leukoencephalopathy, will help in molecular screening for the NOTCH3 gene, thus contributing to enlargement of the NOTCH3 gene variation database.

TARDBP gene mutations in south Italian patients with amyotrophic lateral sclerosis

TAR-DNA-binding protein 43 (TDP-43) has recently been identified as the major pathological protein in abnormal inclusions in neurons and glial cells in sporadic amyotrophic lateral sclerosis (SALS) and SOD1 negative familial cases with amyotrophic lateral sclerosis (FALS). TDP-43 is evolutionarily conserved, consisting of two RNA recognition motifs and a glycine-rich C-terminal domain. It is involved in the regulation of expression and splicing, and in other cellular processes such as microRNA biogenesis, apoptosis and cell division.1 2 Starting in early 2008, dominant mutations in TARDBP gene have been reported by several groups as a primary cause of ALS. To date, a total of 30 mutations of TARDBP have been reported not only in SOD1 -negative FALS cases (∼3%) but also in SALS cases (∼1.5%). All but one of the mutations identified (D169G) reside in exon 6 of the TARDBP gene, which encodes for the C-terminal glycine-rich domain of TDP-43. All of these mutations are dominantly inherited missense changes with the exception of a truncating mutation (Y374X) at the extreme C terminus of the protein.3 In this study, in order to investigate the presence and frequency of TARDBP mutations in a cohort of 310 south Italian patients affected by ALS, we performed a mutational screening of the exon 4 and exon 6 of the gene in SOD1 -negative FALS and SALS patients. …

A novel locus for dHMN with pyramidal features maps to chromosome 4q34.3-q35.2

The distal hereditary motor neuropathy (dHMN) is a rare genetically and clinically heterogeneous disorder characterized by weakness and wasting of distal limb muscles in absence of overt sensory abnormalities. Recently, pyramidal signs have been also described in some patients with dominant or recessive dHMN, and two different loci have been identified in families affected by dHMN complicated with pyramidal dysfunction. We investigated an Italian family affected by an autosomal dominant dHMN complicated by pyramidal signs in order to map a new gene locus. The disease maps to a novel locus in a 26‐cM region flanked by D4S1552 and D4S2930 on chromosome 4q34.3‐35.2. Three candidate genes (SNX25, CASP3 and TUBB4Q) located in the critical region were screened for the presence of mutations by heteroduplex analysis. No mutations have been detected in the analyzed genes. In conclusion, the new private genetic locus we reported further confirms the wide heterogeneity of dHMN.

Neurofibromatosis type 1 & Related Disorders

The last decades have seen major developments in our knowledge of the different forms of neurofibromatosis (Ferner 2007a, b; Ferner et al. 2007; Friedman et al. 1999; Huson and Hughes 1994; Korf and Rubenstein 2005; North 1997; Ruggieri 2007; Upadhyaya and Cooper 1998a). Evidence based clinical diagnostic criteria and management guidelines have been developed (Baser et al. 2002; Ferner et al. 2007; Gutman et al. 1997; Listernick et al. 1997; Listernick and Charrow 2004a, b; North et al. 1997; Maria 2002; Ruggieri 1999; Wolkenstein et al. 1996). The genes for the two major forms, neurofibromatosis type 1 (NF1) and type 2 (NF2), have been cloned and the gene products, neurofibromin and merlin (also called Schwannomin), respectively, fully characterised (reviewed in Baser et al. 2003, Ferner 2007a, Friedman et al. 1999, Korf and Rubenstein 2005, Upadhyaya and Cooper 1998b).

Gene conversion events in adult-onset spinal muscular atrophy

Objective – To investigate the possible occurrence of a conversion event in three patients with adult‐onset spinal muscular atrophy (SMA) type IV, which represents the mildest form within the spectrum of the SMA phenotype.

Abnormally high levels of SOD1 mRNA in a patient with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) occurs in both sporadic (SALS) and, less commonly, familial (FALS) forms. In familial cases, inheritance is usually autosomal dominant, but recessive transmission of the disease also occurs.6 In a subset (15–20%) of FALS cases the disease is caused by mutations in the gene coding Cu–Zn superoxide dismutase (Cu–Zn SOD1) on chromosome 21.5 In SALS cases, mutations have rarely been found. Variability in disease duration exists within families with the same SOD1 mutation. We report a 48-year-old man with a 6-month history of progressive gait difficulty and mild dysarthria. No information was available concerning his father, who died at 40 years of age from an undetermined cause; his mother, aged 74 years, is in good health. Neurologic examination of the patient revealed diffuse fasciculations, brisk tendon reflexes in all limbs, spasticity in the lower limbs, and extensor plantar response bilaterally. Needle electromyography showed fibrillation potentials, positive sharp waves, fasciculations, and increased numbers of polyphasic and long-duration motor unit potentials in the deltoid, dorsal interosseous, vastus medialis, and tibialis anterior muscles on both sides. Motor conduction studies of the right and left median, ulnar, radial, and peroneal nerves were normal, as were sensory studies of the right and left median, ulnar, and sural nerves. Magnetic resonance imaging of the brain and cervical cord were normal. Laboratory evaluation, including thyroid function studies, serum B12 levels, and cerebrospinal fluid examination, was normal. Over the next 10 months, the patient developed dysphagia and his gait difficulties progressed. Examination revealed hyperreflexia, muscular atrophy, and weakness in the arms and legs, and atrophy and fasciculations in his tongue. Sensation was normal. A diagnosis of definite ALS according to the El Escorial criteria7 was made. The patient’s disease progressed rapidly, and he died less than 36 months after onset of symptoms. Molecular investigation, performed with the informed consent of the patient, of DNA samples obtained from peripheral blood, for exon 4 of the SOD1 gene by denaturing high-performance liquid chromatography (DHPLC), revealed a variant band in the patient, and direct sequencing of this polymerase chain reaction product confirmed the occurrence of a deletion of four nucleotides (TACA), 42-bp downstream of the exon–intron splice junction in intron 4. The mutation was not found in 120 control subjects from southern Italy, after they had provided informed consent. Sequence analysis of SOD1 cDNA revealed a normal sequence of the gene. In order to evaluate the impact of this intronic mutation on mRNA production, we performed semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR) by coamplification of the SOD1 gene and glyceraldehyde3-phosphate dehydrogenase (GAPDH) as an endogenous standard sequence. The SOD1 band intensity was stronger in the ALS patient than in normal controls; the ratio SOD1/GAPDH ranged from 1.1 to 1.5 in the ALS patient, but from 0 to 0.8 in normal controls (Fig. 1). This first experimental approach was confirmed, using three normal subjects in duplicate, by the enzyme-linked immunoassay detection assay, a method of quantifying PCR products using a biotin-labeled primer (ELISA kits; Roche, Mannheim, Germany), following the manufacturer’s instructions. This method is more sensitive compared to ethidium bromide–stained gels. We found an intronic deletion in a patient with ALS, associated with high mRNA levels in the patient’s lymphocytes. A similar type of mutation has been documented by Boukaftane et al.,1 who found a deletion in intron 4 of three nucleotides 30-bp downstream of the exon–intron splice junction in a FALS patient, but they did not investigate the mRNA levels. Intronic sequences may play a role in the regulation of the expression of various genes,2,3 and we hypothesize that the observed intronic mutation may have been responsible for the high mRNA level in our patient. Our study indicates that a patient with ALS can have abnormally high levels of SOD1 mRNA. These increased mRNA levels could cause high SOD1 protein concentration, thus increasing SOD1 enzyme activity and leading to an overproduction of reactive oxygen species and neuronal death. Consistent with this hypothesis, Karanjawala et

Further evidence that D90A-SOD1 mutation is recessively inherited in ALS patients in Italy.

Mutations in the Cu/Zn superoxide dismutase 1 (SOD1) gene have been reported to cause adult-onset autosomal dominant amyotrophic lateral sclerosis (FALS). In sporadic cases (SALS), de novo mutations in the SOD1 gene have occasionally been observed. All the SOD1 mutations are autosomal dominantly inherited with the exception of D90A. To date, in Italy, only two sporadic ALS cases carrying the D90A mutation have been reported in a homozygous state. We investigated for the presence of this mutation in 169 unrelated ALS patients from southern Italy. The genetic analysis revealed three ALS patients (1.8%) with mild phenotype carrying the homozygous D90A mutation.

Narrowing of the critical region in autosomal recessive spastic paraplegia linked to the SPG5 locus

Hereditary spastic paraplegias are neurodegenerative disorders characterized clinically by progressive spasticity of the lower limbs; they are inherited as autosomal dominant, autosomal recessive and X‐linked traits. We have analyzed four autosomal recessive HSP families gathered from southern Italy. We performed genetic analysis using microsatellite markers associated with SPG5, SPG7, SPG11 and SPG14. Positive lod scores were obtained with markers located on chromosome 8. The lod scores for the four combined families were significantly higher than 3 for D8S509, D8S1102, D8S1723 and D8S260 with a maximum two‐point lod score at θ = 0 of 3.99 for the marker D8S260. In one of the examined families, the haplotype analysis suggests two key recombination events demonstrating that the gene is localized in the 11 cM region flanked by markers D8S285 and D8S544, refining the ARHSP region by approximately 22 cm. We also analyzed five candidate genes localized within the HSP region: TOX, syndecan‐binding‐protein (SDCBP), RAB2, CA8 and PENK, but we did not find disease causing mutations.

A splice‐junction mutation in SBF2 gene causes autosomal recessive charcot‐marie‐tooth disease (CMT4B2) in a family from southern italy

Autosomal recessive Charcot‐Marie‐Tooth disease type 4 (CMT4) comprises a group of clinically and genetically heterogeneous disorders of the peripheral nervous system. At least 10 loci are responsible for autosomal recessive CMT and six genes have been identified so far. In this study, we report a small pedigree with a recessive form of CMT (CMT4B) from Southern Italy. There were six individuals in two generations with two affected subjects. We performed haplotype analysis using highly polymorphic microsatellite markers located on chromosome 11p15. Subsequently, the coding region of the Sbf2 gene was sequenced by using primers flanking intron‐exon boundaries. Mutational screening of Sbf2 revealed a homozygous mutation in the splice‐junction donor‐acceptor site of intron 32 (+1G→C) in the affected patients. The variation was also confirmed by digestion with restriction enzyme Alu I and it was absent in 100 control chromosomes examined. This is the first finding of a mutation in the Sbf2 gene that alters the correct splicing of the gene. Furthermore, these data confirm that mutations in the Sbf2 gene are causative of CMT4B2.