[Next-generation DNA sequencing in clinical diagnostics]. / Le séquençage d'ADN à haut débit en pratique clinique.

The advent of next generation sequencing (NGS) technologies is so scale-changing that it modifies molecular diagnostics indications and induces laboratories to rethink their diagnostic strategies, until now based on the Sanger sequencing routine. Several high-throughput approaches are available from the sequencing of a gene panel, to an exome, or even a genome. In all cases, a tremendous amount of data is generated, which has to be filtered, interpreted and analyzed using powerful bioinformatics tools. In parallel, ethical considerations are raised to avoid the potential drifts of these powerful approaches. In all medical fields, and particularly in pediatrics, this new strategy offers better efficacy and faster mutation identification, allowing better support and care for patients and their families and even improving genetic counseling. In the present paper, we discuss the different NGS-based approaches and strategies as well as the issues involved in these new technologies applied to molecular diagnosis of rare diseases. Altogether, rare diseases affect more than 3 million people in France and are responsible for about one-third of childhood deaths.

[Distal myopathy due to mutations of GNE gene: clinical spectrum and diagnosis]. / Myopathies distales avec mutations du gène GNE: à propos de quatre cas.

Distal myopathies are rare muscular disorders clinically characterized by a predominantly distal muscular involvement. Among recessive forms, the myopathy resulting from mutations in the UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) gene, often designated as Nonaka myopathy, primarily affect young adults and are characterized by muscle wasting and weakness predominating on the anterior compartment of the leg, a remarkable quadriceps sparing and a frequent evolution towards ambulation loss after a few years. Finding rimmed vacuoles on muscle biopsy is a further argument for the diagnosis. However, the presentation and course may vary and we describe four patients who illustrate the clinical spectrum of the disease: the first patient had a classical form with progressive weakness over several years, the second one a rapidly progressive myopathy leading to ambulation loss within three years from onset, the third one a very slow course with no ambulation loss after several decades, and the last one a progressive form with misleading neurogenic features on the EMG. One of our four patients harbored a homozygous mutation, and three others were compound heterozygous, two of them displaying an original mutation: one had a c.2036 T>G (p.Val679Gly) substitution, the c.829 C>T (p.Arg277Cys) substitution.

Transcriptional explorations of CAPN3 identify novel splicing mutations, a large-sized genomic deletion and evidence for messenger RNA decay.

Mutations in the gene encoding calpain-3 (CAPN3) cause autosomal recessive limb-girdle muscular dystrophy type 2A (LGMD2A) and idiopathic eosinophilic myositis. Accurate diagnosis and genetic counselling are based on the identification of disease-causing mutations on both alleles of CAPN3 in the patients. In the present study, we used transcriptional analysis as a complementary approach for patients suspected of being affected with LGMD2A, in whom initial denaturing high-performance liquid chromatography genomic mutation screening evidenced no or only one CAPN3 mutation obviously considered as disease causing. This allowed to identify and characterize cDNA deletions. Further genomic analysis allowed to determine the origin of these deletions, either as splicing defects caused by intronic mutations or as an internal multi-exonic deletion. In particular, we report two novel CAPN3 mutations (c.1745 + 4_1745 + 7delAGTG in IVS13 and c.2185-16A>G in IVS20) and a recurrent large-sized genomic deletion including exons 2-8 for which genomic breakpoints have been characterized. In addition, our results indicate nonsense-mediated messenger RNA decay as a mechanism for under-expression of CAPN3 associated to some specific variations.

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.

A novel missense mutation (C30S) in the gene encoding tumor necrosis factor receptor 1 linked to autosomal-dominant recurrent fever with localized myositis in a French family.

OBJECTIVE: To characterize both phenotypic (clinical features and magnetic resonance imaging [MRI] findings) and genotypic aspects of autosomal-dominant recurrent fever, also known as tumor necrosis factor receptor (TNFR)-associated periodic syndrome (TRAPS), in a French family and to investigate the role of the mutated 55-kd tumor necrosis factor alpha (TNFalpha) receptor (TNFR1) in the pathogenesis of the disease. METHODS: The coding region of TNFR1 was sequenced in 2 individuals with TRAPS (the propositus and her grandfather) and in 3 clinically unaffected relatives. Expression of soluble TNFR1 (sTNFR1) was investigated in 3 of the family members carrying a C30S mutation in TNFR1, and was compared with the levels of soluble TNFR2 (sTNFR2) by enzyme-linked immunosorbent assay. The membrane TNFR1 expression was then compared with membrane TNFR2 levels at the surface of peripheral blood mononuclear cells by flow cytometric analysis. The clinical heterogeneity in this French family was investigated by searching polymorphic variants in the TNFalpha promoter by DNA sequencing. RESULTS: Both the disease course and the clinical presentation in the propositus were highly indicative of TRAPS. MRI study of the segmental inflammatory process in the limbs showed abnormal signals in the muscle and subcutaneous tissue without involvement of adjacent joints or fascia. A novel missense mutation, C30S, in the first extracellular N-terminal cysteine-rich domain (CRD1) of TNFR1 was characterized in the propositus, her affected grandfather, and her clinically unaffected father. Expression of membrane TNFR1 at the surface of monocytes and polymorphonuclear leukocytes, as well as the levels of sTNFR1 in serum when the disease was not active were not modified in the 3 individuals carrying the TNFR1 C30S mutation. In contrast, during attacks, sTNFR1 levels remained abnormally low, as compared with the levels in unrelated patients with active adult-onset systemic Still's disease. The clinical heterogeneity could not be explained by a polymorphic variant in the TNFalpha promoter. CONCLUSION: TRAPS is a distinct clinical and radiologic disease entity that is responsible for recurrent fever and migratory cellulitis-like processes with localized myositis. We have identified a novel TNFR1 mutation, C30S, that is located in the CRD1 domain in a French family affected by the disease. This mutation seems to affect the level of sTNFR1, which did not increase in the propositus during inflammatory attacks.

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.

Clinical versus genetic diagnosis of familial Mediterranean fever.

The diagnosis of familial Mediterranean fever (FMF) has until recently been based on clinical signs alone. Discovery of the MEFV gene has enabled a molecular approach to diagnosis, which is already well established for diagnosing typical clinical forms of FMF. We evaluated the utility of this molecular approach in a large series of patients with various clinical presentations and ethnic origins. We looked for mutations in the MEFV gene in 303 unselected consecutive patients with a variable (from high to low) clinical suspicion of FMF. Two mutations were found in 133 patients (44%). In 22 patients (7%), the clinical diagnosis of FMF was unlikely according to the Tel Hashomer clinical criteria. Our results suggest that the spectrum of FMF-associated signs is broader than previously believed. Wider indications for genotyping should lead to more frequent diagnosis of FMF.

Renal amyloidosis as a first manifestation of Familial Mediterranean Fever.

Amyloid nephropathy was the presenting symptom in a case of Familial Mediterranean Fever (FMF). As recent progress in molecular pathology permits the detection of asymptomatic FMF individuals, it is suggested that relevant cases of renal amyloidosis should be tested for FMF mutations.

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.

Non-founder mutations in the MEFV gene establish this gene as the cause of familial Mediterranean fever (FMF).

Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by recurring attacks of fever and serositis. It affects primarily North African Jews, Armenians, Turks and Arabs, in which a founder effect has been demonstrated. The marenostrin-pyrin-encoding gene has been proposed as a candidate gene for the disease ( MEFV ), on the basis of the identification of putative mutations clustered in exon 10 (M680V, M694I, M694V and V726A), each segregating with one ancestral haplotype. In a search for additional MEFV mutations in 120 apparently non-founder FMF chromosomes, we observed eight novel mutations in exon 2 (E148Q, E167D and T267I), exon 5 (F479L) and exon 10 (I692del K695R, A744S and R761H). Except for E148Q and K695R, all mutations were found in a single chromosome. Mutation E148Q was found in all ethnic groups studied and in association with a novel ancestral haplotype in non-Ashkenazi Jews (S2). Altogether, these new findings definitively establish the marenostrin/pyrin-encoding gene as the MEFV locus.

Fibrinogen A alpha chain mutation (Arg554 Leu) associated with hereditary renal amyloidosis in a French family.

A French family with hereditary renal amyloidosis (HRA) was studied. The disease presented in 7 of the 8 affected individuals with proteinuria or the nephrotic syndrome. The age of onset was in the fifth decade of life. There is currently no sign of extrarenal involvement in any affected individual. However, the nephropathy in this family is progressive and led to terminal renal failure in 4 patients. Immunohistochemistry studies of glomerular amyloid deposits suggested that the amyloid protein was the fibrinogen A alpha chain. Direct DNA sequencing revealed a G 4993 T transversion and subsequently Arg 554 Leu mutation in the fibrinogen A alpha chain. This is the first description of this fibrinogen A alpha chain mutation in Europe. This family is of French descent and cannot be related to the previously reported Peruvian/Mexican and African-American kindreds.

Sequence analysis of the CAG triplet repeats region in the Huntington disease gene (IT15) in several mammalian species.

Huntington's disease (HD) is a progressive neurodegenerative disease linked to abnormally expanded CAG repeats in the first exon of the IT15 gene. Neither HD or other glutamine expansion triplet disease has not yet been described in other species as human. In this study, we sequenced the region containing the CAG repeat expansion region of the corresponding IT15 gene in several mammalian species. In all investigated species, the number of CAG found is equal or inferior to the normal range of human IT15 alleles. The longer uninterrupted CAG stretch was found in the pig and consists of 18 CAG. In the other species, the CAG stretch was shorter and/or interrupted by other triplets. The proline encoding region following the CAG stretch is conserved in the mammalian species studied here and the number of proline is few or not reduced compared to the number of the glutamine codon repeats region.

Sequence analysis of the CCG polymorphic region adjacent to the CAG triplet repeat of the HD gene in normal and HD chromosomes.

The CAG expansion responsible for Huntington's disease (HD) is followed by an adjacent polymorphic CCG repeat region which may interfere with a PCR based diagnosis. We have sequenced this region in 52 unrelated HD patients, from both normal and HD chromosomes. Fifty percent of the normal alleles were (CCG)7(CCT)2, 48% (CCG)10(CCT)2, and 2% (CCG)7(CCT)3. In contrast (CCG)7(CCT)2 was found in 85% of the HD alleles which represents significant linkage disequilibrium with the HD mutation.

Diagnosis of "sporadic" Huntington's disease.

The diagnosis of Huntington's disease (HD) in patients with progressive chorea and mental impairment, but without similarly affected relatives, remains uncertain and impedes genetic counseling. Twenty patients with suspected HD, but with no family history of the disease underwent molecular analysis of the CAG repeat in the IT15 gene for HD. Eighteen patients displayed the HD expanded allele and two had CAG repeats in the normal range. Neuropsychological tests could be performed in 12 of the 20 patients. Of these 10 with the expanded allele presented the deficits typical of HD, but not the two patients without the HD mutation. This study shows that a neuropsychological pattern is specific to patients with the expanded CAG and that most isolated patients with suspected HD are in fact affected.

Huntington's disease in French families: CAG repeat expansion and linkage disequilibrium analysis.

The molecular defect causing Huntington's disease (HD) has been found as an expansion of CAG triplets in the 5' coding region of IT15 gene. In the 29 French families reported, the HD disease is due to the expansion of the CAG triplets region above 38 copies. The complete sequencing of 10 HD alleles PCR products allowed us to confirm that expansion is restricted to the CAG repeat region and does not extend to the adjacent CCG repeat region which is also present in the PCR product. Then, we analysed linkage disequilibrium between the molecular defect and 6 DNA markers mapping to the 4p16.3 region. The most striking finding in this study is the presence of a strong linkage disequilibrium between HD and D4S127 (PvuII), D4S95 (AccI, MboI, TaqI) located in a region of 130 kb distal to IT15 gene. Two major haplotypes, comprising D4S127 (PvuII) and D4S95 (MboI, AccI) polymorphic sites, were found in the normal population as only one was found associated with HD alleles. This result can be interpreted either as an evidence for a rather recent founder effect or as several independent mutations occuring in chromosomes bearing the same haplotype.