To define the spectrum of mutations in a-, b-, c-, and d-sarcoglycan (SG) genes, we analyzed these genes in 69 probands with clinical and biological criteria compatible with the diagnosis of autosomal recessive limb-girdle muscular dystrophy. For 48 patients, muscle biopsies were available and multiplex western blot analysis of muscle proteins showed significant abnormalities of a-and c-SG. Our diagnostic strategy includes multiplex western blot, sequencing of SG genes, multiplex quantitative-fluorescent PCR and RT-PCR analyses. Mutations were detected in 57 patients and homozygous or compound heterozygous mutations were identified in 75% (36/48) of the patients with abnormal western blot, and in 52% (11/21) of the patients without muscle biopsy. Involvement of a-SG was demonstrated in 55.3% of cases (26/47), whereas c-and b-SG were implicated in 25.5% (12/47) and in 17% (8/47) of cases, respectively. Interestingly, we identified 25 novel mutations, and a significant proportion of these mutations correspond to deletions (identified in 14 patients) of complete exon(s) of a-or c-SG genes, and partial duplications (identified in 5 patients) of exon 1 of b-SG gene. This study highlights the high frequency of exonic deletions of a-and c-SG genes, as well as the presence of a hotspot of duplications affecting exon 1 of the b-SG gene. In addition, protein analysis by multiplex western blot in combination with mutation screening and genotyping results allowed to propose a comprehensive and efficient diagnostic strategy and strongly suggested the implication of additional genes, yet to be identified, in sarcoglycanopathy-like disorders.

Summary Pyruvate kinase (PK) deficiency is the most common enzyme defect affecting the glycolytic pathway of the erythrocyte. Usually, it is clinically silent in heterozygotes but serious disorders are described at birth in homozygotes or compound heterozygotes. Including the mutants herein reported, more than 180 mutations of the PK‐LR gene have now been identified. This 3‐year study was carried out to detect mutations associated with disease‐affecting families. Haematological indices, erythrocyte PK and glucose‐6‐phosphate dehydrogenase activities were measured. Molecular characterisation of the PK gene mutations included restriction enzyme analysis, mutation scanning and gene sequencing. Among the 56 families studied, nine homozygous cases and 41 different mutations were found. Eight mutations involved a splice site, 31 missense mutations were located in crucial domains of the molecule (catalytic site, cleft between the A and C domains, A/A’ interface) and two cases of insertion–deletion were found. In total, 20 new mutations modifying the structure of the enzyme and seven affecting a splice site are reported. PK deficiency is an under diagnosed disease. However, deficiency could be life threatening in perinatal period and we report two lethal cases. These results support the characterisation of PK mutations, and show that prenatal diagnosis can identify affected infants and prepare safer conditions for the birth.

For the Focus Section on Array-CGHGenomic copy-number variations (CNVs) involving large DNA segments are known to cause many genetic disorders. Depending on the changes, they are predicted to lead either to decreased or an increased gene expression. However, the ability to detect smaller exonic copy-number changes has not been explored. Here we describe a new oligonucleotide-based comparative genomic hybridization (CGH)-array approach for highthroughput detection of exonic deletions or duplications and its application to deletion/duplication analyses of the genes encoding CFTR, six sarcoglycans (SGCA, SGCB, SGCG, SGCD, SGCE, and SGCZ), and DMD. In this work we show the successful development of an array format containing 158 exons that collectively span eight genes and its clinical application for the rapid screening of deletions and duplications in a diagnostic setting. We have analyzed a series of 35 DNA samples from patients affected with cystic fibrosis (CF), Duchenne and Becker muscular dystrophies (DMD/BMD), or sarcoglycanopathies, and have characterized exonic copy-number changes that have been validated with other methods. Interestingly, even heterozygous deletions and duplications of only one exon, as well as mosaic deletions, were detected by this CGH approach. Our results showed that the resolution is very high, as abnormalities of about 1.5-2 kb could be detected. Since this approach is completely scalable, this new molecular tool will allow the screening of combinations of genes involved in a particular group of clinically and genetically heterogeneous disorders such as mental retardation, muscular dystrophies and brain malformations. Hum Mutat 29(9), [1083][1084][1085][1086][1087][1088][1089][1090] 2008.

The frequency of disease-related large rearrangements (referred to as copy-number mutations, CNMs) varies among genes, and search for these mutations has an important place in diagnostic strategies. In recent years, CGH method using custom-designed high-density oligonucleotide-based arrays allowed the development of a powerful tool for detection of alterations at the level of exons and made it possible to provide flexibility through the possibility of modeling chips. The aim of our study was to test custom-designed oligonucleotide CGH array in a diagnostic laboratory setting that analyses several genes involved in various genetic diseases, and to compare it with conventional strategies. To this end, we designed a 12-plex CGH array (135k; 135 000 probes/subarray) (Roche Nimblegen) with exonic and intronic oligonucleotide probes covering 26 genes routinely analyzed in the laboratory. We tested control samples with known CNMs and patients for whom genetic causes underlying their disorders were unknown. The contribution of this technique is undeniable. Indeed, it appeared reproducible, reliable and sensitive enough to detect heterozygous single-exon deletions or duplications, complex rearrangements and somatic mosaicism. In addition, it improves reliability of CNM detection and allows determination of boundaries precisely enough to direct targeted sequencing of breakpoints. All of these points, associated with the possibility of a simultaneous analysis of several genes and scalability ‘homemade' make it a valuable tool as a new diagnostic approach of CNMs.

Our results illustrate, in a large series of patients, the important role of RE and other genomic features in DNA breaks, and the involvement of different mechanisms in DMD gene deletions: Mainly replication error repair mechanisms, but also NHEJ and potentially aberrant firing of replication origins. A combination of these mechanisms may also be possible.

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Homozygous or compound heterozygous for Pyruvate Kinase (PK) deficiency are classical etiology for chronic non spherocytic hemolytic anemias while heterozygous carriers are free from disease. We report here 2 patients heterozygous for PK deficiency which displayed an unexpected marked chronic anemia. Enzymatic and molecular studies were performed to unravel the mechanism causing this phenotype. The first patient, a 60-year-old woman from Mali presented with Hb 10g/dL, MCV 98fL, and was free from any Hb abnormality. The second one was the first child of a healthy French Caucasian couple, and suffered since birth from a marked hemolytic anemia (Hb 7g/dL). We found that the first patient carried together an Arg569Met/Leu PK-R mutation (Pissard et al. Brit J Haematol, 2006, 133, 683-9) and the rare G6PD Santa Maria mutation (nt c.542 A>T, Asp181Val). The second patient had a PK splice site mutation (IVS4 + 10 G>T) and a new hexokinase mutation [c.1793_c.1836 +7(del 50)] which starts in exon 12 and ends in intron 12. It results in a protein troncated inside the glucose binding site. In this case, family study showed that the PK deficiency was inherited from the father and the HK deficiency from the mother. Enzymatic data are shown in the table. None of these enzymatic defects could alone, in the heterozygous state, be responsible for an hemolytic anemia. To explain why, in these two cases the combination of two defects resulted in a hemolytic disease, we hypothesized that the increase of the intra-erythrocytic 2,3-DPG level resulting from the PK-R deficiency might cause these disorders. It is well known that increase of the 2,3-DPG level dramatically change several properties of the RBCs such as a decrease in oxygen affinity and an inhibition of the G6PD activity (Tomoda A. Brit J Haematol, 1983, 54, 475 – 84). It has been shown that, when associated to a sickle cell trait, PK-R deficiency by increasing 2,3-DPG leads to sickle cell anemia (Cohen.Solal M. et al, Brit J Haematol, 1998, 103, 950-6). We propose that, in these two patients, the global mechanism leading to the disease results from the increased 2,3-DPG which cause a failure in the anti oxidant pathway. In the first case G6PD inhibition occurs along with a mutated enzyme and in the second one inhibition take place in a under supplied pentose phosphate pathway due to the hexokinase deficiency. Together with the diminished ATP supply of the cell, this decreased anti-oxidant activity might cause the hemolysis. Thus in any anemic heterozygous PK deficient patient, another RBC abnormality needs to be searched for. enzymatic data patients Pk activity (5.9–8.1) g6pd activity (5.3–7.9) hexokinase activity (0.74–1.14) 2.3 DPG (11.7–15.3) nd : not determined 1 3.2 UI/g Hb 4.5 UI/g Hb nd 21.2 μM /g Hb 2 6.8 UI /g Hb 9.2 UI /g Hb 0.3 UI / g Hb 36.8 μM /g Hb