Unravelling the etiology of sporadic late-onset cerebellar ataxia in a cohort of 205 patients: a prospective study.

BACKGROUND: Despite recent progress in the field of genetics, sporadic late-onset (> 40 years) cerebellar ataxia (SLOCA) etiology remains frequently elusive, while the optimal diagnostic workup still needs to be determined. We aimed to comprehensively describe the causes of SLOCA and to discuss the relevance of the investigations. METHODS: We included 205 consecutive patients with SLOCA seen in our referral center. Patients were prospectively investigated using exhaustive clinical assessment, biochemical, genetic, electrophysiological, and imaging explorations. RESULTS: We established a diagnosis in 135 (66%) patients and reported 26 different causes for SLOCA, the most frequent being multiple system atrophy cerebellar type (MSA-C) (41%). Fifty-one patients (25%) had various causes of SLOCA including immune-mediated diseases such as multiple sclerosis or anti-GAD antibody-mediated ataxia; and other causes, such as alcoholic cerebellar degeneration, superficial siderosis, or Creutzfeldt-Jakob disease. We also identified 11 genetic causes in 20 patients, including SPG7 (n = 4), RFC1-associated CANVAS (n = 3), SLC20A2 (n = 3), very-late-onset Friedreich's ataxia (n = 2), FXTAS (n = 2), SCA3 (n = 1), SCA17 (n = 1), DRPLA (n = 1), MYORG (n = 1), MELAS (n = 1), and a mitochondriopathy (n = 1) that were less severe than MSA-C (p < 0.001). Remaining patients (34%) had idiopathic late-onset cerebellar ataxia which was less severe than MSA-C (p < 0.01). CONCLUSION: Our prospective study provides an exhaustive picture of the etiology of SLOCA and clues regarding yield of investigations and diagnostic workup. Based on our observations, we established a diagnostic algorithm for SLOCA.

Deciphering the causes of sporadic late-onset cerebellar ataxias: a prospective study with implications for diagnostic work.

The management of sporadic late-onset cerebellar ataxias represents a very heterogeneous group of patients and remains a challenge for neurologist in clinical practice. We aimed at describing the different causes of sporadic late-onset cerebellar ataxias that were diagnosed following standardized, exhaustive investigations and the population characteristics according to the aetiologies as well as at evaluating the relevance of these investigations. All patients consecutively referred to our centre due to sporadic, progressive cerebellar ataxia occurring after 40 years of age were included in the prospective, observational study. 80 patients were included over a 2 year period. A diagnosis was established for 52 patients (65%) corresponding to 18 distinct causes, the most frequent being cerebellar variant of multiple system atrophy (n = 29). The second most frequent cause was inherited diseases (including spinocerebellar ataxias, late-onset Friedreich's disease, SLC20A2 mutations, FXTAS, MELAS, and other mitochondrial diseases) (n = 9), followed by immune-mediated or other acquired causes. The group of patient without diagnosis showed a slower worsening of ataxia (p < 0.05) than patients with multiple system atrophy. Patients with later age at onset experienced faster progression of ataxia (p = 0.001) and more frequently parkinsonism (p < 0.05) than patients with earlier onset. Brain MRI, DaT scan, genetic analysis and to some extent muscle biopsy, thoracic-abdominal-pelvic tomodensitometry, and cerebrospinal fluid analysis were the most relevant investigations to explore sporadic late-onset cerebellar ataxia. Sporadic late-onset cerebellar ataxias should be exhaustively investigated to identify the underlying causes that are numerous, including inherited causes, but dominated by multiple system atrophy.

[Melanoma within naevus spilus: 5 cases]. / Mélanome sur naevus spilus: 5 observations.

BACKGROUND: Nevus spilus is defined as café-au-lait macules with dark maculopapular speckles. Histologically, it has the aspect of lentigo associated with nevocellular nevus. There are 3 types of nevus spilus: small or medium-sized (<20 cm), giant and zosteriform. Malignant transformation of nevus spilus is rare. PATIENTS AND METHODS: We analyzed the cases of 5 patients presenting melanoma within nevus spilus as well as 20 published cases. The evaluation criteria were: for nevus spilus: size, type, topography, age of onset and presence of dysplastic nevi within the nevus spilus; for melanoma: clinical aspect, histological type, thickness, level and age at diagnosis. The presence of other risk factors for melanoma was noted. RESULTS: The 14 women and 11 men had a mean age of 49 years at melanoma diagnosis. Type of nevus spilus was: small or medium-sized (15 cases), zosteriform (6 cases) and giant (4 cases). Only 3 nevi spili were<4 cm in diameter. Nevus spilus was present since birth (11 cases), childhood (7 cases), after the age of 20 years (3 cases) and was unspecified in 4 cases. Three of our five patients had other risk factors for melanoma. Two patients were presenting 2 melanomas within nevus spilus. The histological type of melanoma was not specified in 8 cases but SSM was the most common type (13 cases). Median Breslow thickness was 1.25 mm (0.27 to 8 mm) for the 19 cases in which it was specified. CONCLUSION: The following criteria appeared to be associated with risk of developing melanoma in nevus spilus patients: nevus spilus present since birth, nevus spilus over 4 cm in diameter, and giant or zosteriform nevus spilus. Development of melanoma within nevus spilus is a rare event. Consequently, guidelines for follow-up of nevus spilus cannot be defined. However, follow-up is recommended, and in particular, self-examination.

Comprehensive cystic fibrosis mutation epidemiology and haplotype characterization in a southern Italian population.

We screened the whole coding region of the cystic fibrosis transmembrane regulator (CFTR) gene in 371 unrelated cystic fibrosis (CF) patients from three regions of southern Italy. Forty-three mutations detected 91.5% of CF mutated chromosomes by denaturing gradient gel electrophoresis analysis, and three intragenic CFTR polymorphisms predicted a myriad of rare mutations in uncharacterized CF chromosomes. Twelve mutations are peculiar to CF chromosomes from southern Italy: R1158X, 4016insT, L1065P and 711 + 1G > T are present in 6.3% of CF chromosomes in Campania; G1244E and 852del22 are present in 9.6% of CF chromosomes in Basilicata and 4382delA, 1259insA, I502T, 852del22, 4016insT, D579G, R1158X, L1077P and G1349D are frequent in Puglia (19.6% of CF alleles). Several mutations frequently found in northern Italy (e.g., R1162X, 711 + 5G > T) and northern Europe (e.g., G551D, I507del and 621 + 1G > T) are absent from the studied population. The I148T-3195del6 complex allele was present in two CF chromosomes, whereas I148T was present in both alleles (as a single mutation) in another CF patient and in five CF carriers; this could result from crossover events. The haplotype analysis of three intragenic polymorphisms (IVS8CA, IVS17bTA and IVS17bCA) compared with data from other studies revealed that several mutations (3849 + 10kbC > T, 1717-1G > A, E585X, 3272-26G > A, L558S, 2184insA and R347P) originated from multiple events, whereas others (R1158X and S549R) could be associated with one or more intragenic recombinant events. Given the large population migration from southern Italy, knowledge of the CF molecular epidemiology in this area is an important contribution to diagnosis, counselling and interlaboratory quality control for molecular laboratories worldwide.

Familial Mediterranean fever in Lebanon: mutation spectrum, evidence for cases in Maronites, Greek orthodoxes, Greek catholics, Syriacs and Chiites and for an association between amyloidosis and M694V and M694I mutations.

Seventy-nine unrelated Lebanese patients were tested for 15 mutations in the MEFV gene: A761H, A744S, V726A, K695R, M694V, M694I, M694del, M6801 (G --> C), M680I (G --> A) in exon 10, F479L in exon 5, P369S in exon 3, T267I, E167D and E148Q in exon 2, using PCR digestion, ARMS, DGGE and/or sequencing. Mutations were detected in patients belonging to all communities, most interestingly the Maronite, Greek orthodox, Greek catholic, Syriac and Chiite communities. The most frequent mutations are M694V and V726A (27% and 20% of the total alleles respectively). M694I, E148Q and M680I mutations account respectively for 9%, 8% and 5%. Each of the K695R, E167D and F479L mutations was observed once and all the remaining mutations were not encountered. Of the alleles 33% do not carry any of the studied mutations. The mutation spectra, clinical features and severity of the disease differed among the Lebanese communities. The genotype-phenotype analysis showed a significant association (P < 0.001) between amyloidosis and the presence of mutations at codon 694 in exon 10 (both M694V and M694I). None of the patients carrying other mutations developed amyloidosis.

Identification of MEFV-independent modifying genetic factors for familial Mediterranean fever.

Familial Mediterranean fever (FMF) is a recessively inherited disorder predisposing to renal amyloidosis and associated with mutations in MEFV, a gene encoding a protein of unknown function. Differences in clinical expression have been attributed to MEFV-allelic heterogeneity, with the M694V/M694V genotype associated with a high prevalence of renal amyloidosis. However, the variable risk for patients with identical MEFV mutations to develop this severe complication, prevented by lifelong administration of colchicine, strongly suggests a role for other genetic and/or environmental factors. To overcome the well-known difficulties in the identification of modifying genetic factors, we investigated a relatively homogeneous population sample consisting of 137 Armenian patients with FMF from 127 independent families living in Armenia. We selected the SAA1, SAA2, and APOE genes-encoding serum amyloid proteins and apolipoprotein E, respectively-as well as the patients' sex, as candidate modifiers for renal amyloidosis. A stepwise logistic-regression analysis showed that the SAA1alpha/alpha genotype was associated with a sevenfold increased risk for renal amyloidosis, compared with other SAA1 genotypes (odds ratio [OR] 6. 9; 95% confidence interval [CI] 2.5-19.0). This association, which was present whatever the MEFV genotype, was extremely marked in patients homozygous for M694V (11/11). The risk for male patients of developing renal amyloidosis was fourfold higher than that for female patients (OR=4.0; 95% CI=1.5-10.8). This association, particularly marked in patients who were not homozygous for M694V (34.0% vs. 11.6%), was independent of SAA1-allelic variations. Polymorphisms in the SAA2 or APOE gene did not appear to influence susceptibility to renal amyloidosis. Overall, these data, which provide new insights into the pathophysiology of FMF, demonstrate that susceptibility to renal amyloidosis in this Mendelian disorder is influenced by at least two MEFV-independent factors of genetic origin-SAA1 and sex-that act independently of each other.

NF1 gene analysis focused on CpG-rich exons in a cohort of 93 patients with neurofibromatosis type 1.

We studied the NF1 gene in 93 unrelated patients with neurofibromatosis type1, focusing the analysis on four exons that contain the highest number of possible mutations occurring at CpG sites. We used denaturing gradient gel electrophoresis to analyse exons 16, 28, 29 and 49, which contain 45 (25%) of the 183 possible mutations that could occur at the 120 CpG dinucleotides of the coding sequence. Six different mutations were identified, five of which are novel: two truncating mutations, W1810X and 5448insG, located in exon29; two splice defects leading to exon29 skipping, 5206-2A>G and 5546G>A; and one missense mutation, L844F, located in exon16. The already described R1748X mutation located in exon29 was found in two unrelated patients. The 5546G>A and R1748X mutations are located at CpG sites, whereas the W1810X involves a CpNpG site. Four novel polymorphisms, which may be helpful for family studies, were also identified. Overall, all but one mutations were found in exon29, a result which suggests that all the CpG sites of the NF1 coding sequence do not have the same mutability, and that exon29, the most CpG-rich exon, contains mutational hotspots associated with NF1.

Spectrum of CFTR mutations in cystic fibrosis and in congenital absence of the vas deferens in France.

We have collated the results of cystic fibrosis (CF) mutation analysis conducted in 19 laboratories in France. We have analyzed 7, 420 CF alleles, demonstrating a total of 310 different mutations including 24 not reported previously, accounting for 93.56% of CF genes. The most common were F508del (67.18%; range 61-80), G542X (2.86%; range 1-6.7%), N1303K (2.10%; range 0.75-4.6%), and 1717-1G>A (1.31%; range 0-2.8%). Only 11 mutations had relative frequencies >0. 4%, 140 mutations were found on a small number of CF alleles (from 29 to two), and 154 were unique. These data show a clear geographical and/or ethnic variation in the distribution of the most common CF mutations. This spectrum of CF mutations, the largest ever reported in one country, has generated 481 different genotypes. We also investigated a cohort of 800 French men with congenital bilateral absence of the vas deferens (CBAVD) and identified a total of 137 different CFTR mutations. Screening for the most common CF defects in addition to assessment for IVS8-5T allowed us to detect two mutations in 47.63% and one in 24.63% of CBAVD patients. In a subset of 327 CBAVD men who were more extensively investigated through the scanning of coding/flanking sequences, 516 of 654 (78. 90%) alleles were identified, with 15.90% and 70.95% of patients carrying one or two mutations, respectively, and only 13.15% without any detectable CFTR abnormality. The distribution of genotypes, classified according to the expected effect of their mutations on CFTR protein, clearly differed between both populations. CF patients had two severe mutations (87.77%) or one severe and one mild/variable mutation (11.33%), whereas CBAVD men had either a severe and a mild/variable (87.89%) or two mild/variable (11.57%) mutations.

Mutations in the MEFV gene in a large series of patients with a clinical diagnosis of familial Mediterranean fever.

Familial Mediterranean fever (FMF) is an autosomal recessively inherited disease affecting patients of the Mediterranean basin. FMF is characterized by recurrent episodes of fever accompanied with topical signs of inflammation. Some patients can develop a renal amyloidosis associated (AA) amyloidosis. The administration of colchicine is an effective preventive treatment of both the attacks and amyloidosis. The FMF gene (MEFV) was cloned and missense mutations were found to be responsible for the disease. We investigated a large series of 303 unselected and unrelated patients of various ethnic backgrounds with a clinical suspicion of FMF to confirm or invalidate the diagnosis of FMF and to determine the spectrum of MEFV mutations. Molecular analysis focused on all the most frequent mutations identified so far, and an exhaustive analysis of exon 10, containing the mutational hotspot, was performed through DNA sequencing. Sixty-two percent of Sephardic, North African Arabs, Armenian and Turkish patients were either homozygous or compound heterozygous for MEFV mutations. In other populations surrounding the Mediterranean Sea such as Greek, Italian, Portuguese, Kurdish and Lebanese populations, mutations were also found. In general, patients without Mediterranean origin had no mutations in the MEFV gene. Two new mis-sense mutations were identified in exon 10 of the MEFV gene: the S675N in an Italian patient and the M680L in a French patient without any known at-risk ethnic ancestry.

Screening practices for mutations in the CFTR gene ABCC7.

Cystic fibrosis transmembrane conductance regulator (CFTR) gene studies are now one of the most frequent activities in clinical molecular genetics laboratories. The number of requests is growing, owing to the increasingly wide range of recognized CFTR gene diseases (cystic fibrosis, congenital bilateral absence of the vas deferens, disseminated bronchiectasis, allergic bronchopulmonary aspergillosis and chronic pancreatitis), and the availability of efficient molecular tools for detecting mutations. A growing number of tests capable of simultaneously detecting several frequent CF mutations are being developed, and commercial kits are now available. The most recent kits detect nearly 90% of defective alleles in Caucasians, a rate high enough for carrier screening and for the majority of diagnostic requests. However, because of the wide variety of molecular defects documented in the CFTR gene, only a limited number of laboratories have mastered the entire panoply of necessary techniques, while other laboratories have to refer certain cases to specialized centers with complementary and/or scanning tools at their disposal. A good knowledge of CFTR diseases and their molecular mechanisms, together with expertise in the various techniques, is crucial for interpreting the results. Diagnostic strategies must take into account the indication, the patient's ethnic origin, and the time available in the framework of genetic counseling. This review presents the methods most frequently used for detecting CFTR gene mutations, and discusses the strategies most suited to the different clinical settings.

Liver iron accumulation in patients with chronic active hepatitis C: prevalence and role of hemochromatosis gene mutations and relationship with hepatic histological lesions.

BACKGROUND/AIMS: Liver iron accumulation has been described in patients with chronic active hepatitis (CAH) C, and could play a role in the course of liver disease and negatively influence the response to interferon. The aim of this study was to determine the prevalence and severity of liver iron accumulation in CAH C, to assess its relationship with the HFE C282Y and H63D mutations, and to study its interactions with hepatic histological lesions. METHODS: Two hundred and nine patients (131 men, 78 women, mean age 44.3+/-12.0 years) with CAH C, including 19 patients with cirrhosis (9.1%) were studied. A semiquantitative grading system from 0 to 3 was used for histological assessment of liver iron accumulation on Perls' staining. The HFE C282Y and H63D mutations were screened for by restriction enzyme analysis performed on PCR-amplified products. Histological scores of activity and fibrosis were determined according to a previously validated METAVIR score system. RESULTS: Liver iron accumulation was found in 88/209 patients (42.1%), and was generally mild. The C282Y and H63D allele frequencies were in 23 (11.0%), and 50 (23.9%), respectively. No association was found between the presence of liver iron accumulation and the detection of the C282Y and H63D mutations. A significant relationship was found between the severity of histological activity and liver iron accumulation of macrophagic or mixed (i.e. both macrophagic and hepatocytic) type (p = 0.04). Although the number of cirrhotic patients was small, cirrhosis was more frequently observed in patients with than without liver iron accumulation (17.2% vs. 3.3%, p = 0.004). CONCLUSIONS: Overall, these data suggest that the liver iron accumulation in patients with CAH C is significantly associated with histological activity and cirrhosis, whereas the two missense hemochromatosis gene mutations are not major determinants.

Detection of five rare cystic fibrosis mutations peculiar to Southern Italy: implications in screening for the disease and phenotype characterization for patients with homozygote mutations.

BACKGROUND: The search for the eight most frequent mutations (i.e., DeltaF508, G542X, W1282X, N1303K, 1717-1G-->A, R553X, 2183AA-->G, and I148T) by allele-specific oligonucleotide dot-blot analysis revealed 78% of 396 cystic fibrosis alleles in Southern Italy. The observation of frequent haplotypes on the unidentified cystic fibrosis alleles suggested that a few mutations could account for a large number of unidentified alleles. METHODS: We screened most of the coding sequence of the cystic fibrosis transmembrane regulator gene by denaturing gradient gel electrophoresis to determine the spectrum of these mutations in 68 unrelated cystic fibrosis patients bearing one or both unidentified mutations. RESULTS: The screening revealed five mutations, R1158X, 711+1G-->T, 4016insT, L1065P, and G1244E, each of which had a frequency of 1.3-1.8% (7% collectively). The 7% increase in the detection rate (85% vs 78%) reduces by >50% the residual risk of being cystic fibrosis carriers for couples who had tested negative by molecular analysis. We therefore designed a second allele-specific oligonucleotide set to analyze the five mutations. Among the patients analyzed, one patient homozygous for the L1065P mutation expressed a mild pulmonary and intestinal form of the disease with pancreatic insufficiency. Two other patients, homozygous for mutations R1158X and 4016insT, both expressed a severe cystic fibrosis phenotype. CONCLUSIONS: Five cystic fibrosis mutations are peculiar to patients from Southern Italy. The method described for their analysis is efficient, inexpensive, and can be semi-automated by use of a robotic workstation. The results obtained in patients from Southern Italy may have an impact on laboratories in other countries, given the large migrations of populations from Southern Italy to other countries in the last two centuries.

MEFV-Gene analysis in armenian patients with Familial Mediterranean fever: diagnostic value and unfavorable renal prognosis of the M694V homozygous genotype-genetic and therapeutic implications.

Familial Mediterranean fever (FMF) is a recessively inherited disorder that is common in patients of Armenian ancestry. To date, its diagnosis, which can be made only retrospectively, is one of exclusion, based entirely on nonspecific clinical signs that result from serosal inflammation and that may lead to unnecessary surgery. Renal amyloidosis, prevented by colchicine, is the most severe complication of FMF, a disorder associated with mutations in the MEFV gene. To evaluate the diagnostic and prognostic value of MEFV-gene analysis, we investigated 90 Armenian FMF patients from 77 unrelated families that were not selected through genetic-linkage analysis. Eight mutations, one of which (R408Q) is new, were found to account for 93% of the 163 independent FMF alleles, with both FMF alleles identified in 89% of the patients. In several instances, family studies provided molecular evidence for pseudodominant transmission and incomplete penetrance of the disease phenotype. The M694V homozygous genotype was found to be associated with a higher prevalence of renal amyloidosis and arthritis, compared with other genotypes (P=.0002 and P=.006, respectively). The demonstration of both the diagnostic and prognostic value of MEFV analysis and particular modes of inheritance should lead to new ways for management of FMF-including genetic counseling and therapeutic decisions in affected families.

CFTR gene mutations in adults with disseminated bronchiectasis.

The severity and type of clinical manifestations are variable in patients with cystic fibrosis (CF). The respiratory syndromes in these patients consist of lung infections associated with disseminated bronchiectasis (DB), asthma, and chronic obstructive pulmonary disease. To investigate the possible involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in chronic pulmonary disease in adults, we studied 32 DB patients with a clinically isolated respiratory syndrome. Careful analysis of all the CFTR gene exons and their flanking regions revealed a significantly increased frequency of CFTR gene mutations in these patients. Thirteen CFTR gene mutations were identified in sixteen different alleles. Six of these mutations, which have previously been reported as CF defects, were found on nine alleles. A further four, two of which had not previously been described (D192N and 406-2 AdeltaC), are potentially disease-causing mutations. We also identified three rare substitutions (R31C, L997F, T1220I), which could be involved in mild CFTR gene disease. Four patients were compound heterozygotes, one carried two CFTR gene mutations (possibly allelic) and six were heterozygous for a mutation. These results indicate that CFTR gene mutations may play a role in bronchiectatic lung disease, possibly in a multifactorial context. These findings have implications for genetic counselling of DB patients and their families.

Three novel sequence variations in the 5' upstream region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene: two polymorphisms and one putative molecular defect.

More than 400 sequence alterations have been identified in the whole coding sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene corresponding to the 27 exons and their exon-intron boundaries. However, in some CF chromosomes, no mutation has yet been detected. In such cases, we have explored the promoter and the sequence up to position -1000 from the cap site, by using denaturing gradient gel electrophoresis. This study concerning 35 CF chromosomes has allowed us to identify three novel sequence variations located in the 5' upstream region of the gene. The T to G substitution located at position -895 from the cap site could be considered as a polymorphic variation. The second substitution (C to T at position -816) has been detected on only one CF chromosome, but does not concern a regulatory DNA element previously described. Conversely, the third substitution (a T to G substitution at position -741 from the cap site) is located at a potential AP-1 binding site. We have investigated, by electrophoretic mobility shift assay, the ability of this region to bind nuclear factors. We have found that the normal sequence between -740/-745 does not bind either the AP-1 transcription factor or AP-1 related proteins, and that the T to G-741 mutated sequence exhibits an abnormal binding pattern suggesting the possible deleterious effect of still unknown negative trans-acting factors.

Mutation heterogeneity of cystic fibrosis in France: screening by denaturing gradient gel electrophoresis using psoralen-modified oligonucleotide.

In order to establish counseling guidelines and aid carrier risk assessment, we sought to establish the frequencies of cystic fibrosis (CF) mutations that are present in CF families living in the Ile de France region, a region notable for its ethnic heterogeneity. We studied 470 CF chromosomes in which we identified more than 90% of the CF mutations. We systematically screened 21 exons and the adjacent sequences of the CF transmembrane conductance regulator (CFTR) gene by denaturing gradient gel electrophoresis using chemical clamps. We detected 41 different CF mutations located in 14 exons. One of these mutations had not been previously described. Given the heterogeneity of these mutations, population screening does not appear to be readily feasible in our population.