Genomic characterization of large rearrangements of the LDLR gene in Czech patients with familial hypercholesterolemia.

BACKGROUND: Mutations in the LDLR gene are the most frequent cause of Familial hypercholesterolemia, an autosomal dominant disease characterised by elevated concentrations of LDL in blood plasma. In many populations, large genomic rearrangements account for approximately 10% of mutations in the LDLR gene. METHODS: DNA diagnostics of large genomic rearrangements was based on Multiple Ligation dependent Probe Amplification (MLPA). Subsequent analyses of deletion and duplication breakpoints were performed using long-range PCR, PCR, and DNA sequencing. RESULTS: In set of 1441 unrelated FH patients, large genomic rearrangements were found in 37 probands. Eight different types of rearrangements were detected, from them 6 types were novel, not described so far. In all rearrangements, we characterized their exact extent and breakpoint sequences. CONCLUSIONS: Sequence analysis of deletion and duplication breakpoints indicates that intrachromatid non-allelic homologous recombination (NAHR) between Alu elements is involved in 6 events, while a non-homologous end joining (NHEJ) is implicated in 2 rearrangements. Our study thus describes for the first time NHEJ as a mechanism involved in genomic rearrangements in the LDLR gene.

Fragment analysis represents a suitable approach for the detection of hotspot c.7541_7542delCT NOTCH1 mutation in chronic lymphocytic leukemia.

The hotspot c.7541_7542delCT NOTCH1 mutation has been proven to have a negative clinical impact in chronic lymphocytic leukemia (CLL). However, an optimal method for its detection has not yet been specified. The aim of our study was to examine the presence of the NOTCH1 mutation in CLL using three commonly used molecular methods. Sanger sequencing, fragment analysis and allele-specific PCR were compared in the detection of the c.7541_7542delCT NOTCH1 mutation in 201 CLL patients. In 7 patients with inconclusive mutational analysis results, the presence of the NOTCH1 mutation was also confirmed using ultra-deep next generation sequencing. The NOTCH1 mutation was detected in 15% (30/201) of examined patients. Only fragment analysis was able to identify all 30 NOTCH1-mutated patients. Sanger sequencing and allele-specific PCR showed a lower detection efficiency, determining 93% (28/30) and 80% (24/30) of the present NOTCH1 mutations, respectively. Considering these three most commonly used methodologies for c.7541_7542delCT NOTCH1 mutation screening in CLL, we defined fragment analysis as the most suitable approach for detecting the hotspot NOTCH1 mutation.

Analysis of histopathologic and molecular pathologic findings in Czech LGMD2A patients.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal-recessive disorder characterized by selective atrophy and progressive weakness of proximal girdle muscles. LGMD2A, the most prevalent form of LGMD, is caused by mutations in the CAPN3 gene that encodes the skeletal muscle-specific member of the calpain family, calpain-3 (p 94). We examined the histopathologic and molecular pathologic findings in 14 Czech LGMD2A patients. Analysis of the CAPN3 gene was performed at the mRNA level, using reverse transcription-polymerase chain reaction (RT-PCR) and sequencing, and/or DNA level, using PCR and denaturing high-performance liquid chromatography (DHPLC). Our results confirm that mutation 550 delA is the most frequent CAPN3 defect in Czech LGMD2A patients (9 alleles of 28). Furthermore, we established that, in a patient with the 550 delA/R490W genotype, mRNA carrying frameshift mutation 550 delA was not detected, probably due to its degradation by nonsense-mediated mRNA decay. In muscle biopsies of two LGMD2A patients, a neurogenic pattern simulating a neurogenic lesion was observed. Immunoblot analysis revealed the deficiency of p 94 in all genetically confirmed cases of LGMD2A, and secondary dysferlin deficiency was demonstrated on muscle membranes in 6 patients using immunofluorescence. Thus, we find a combination of DNA and mRNA mutational analysis to be useful in the diagnosis of LGMD2A. Moreover, our study expands the spectrum of calpainopathies to cases that simulate a neurogenic lesion in muscle biopsies, and the knowledge of possible secondary deficiencies of muscular proteins also contributes to a diagnosis of LGMD2A.

Mutation analysis of the ATP7B gene and genotype/phenotype correlation in 227 patients with Wilson disease.

Wilson disease (WD) is an autosomal recessive disorder of copper transport. WD patients are presenting with a wide range of heterogeneous clinical syndromes including hepatic, neurological, or psychiatric presentations. The disease is caused by mutations in the ATP7B gene. This study presents the results of comprehensive mutation analysis in 227 WD patients from 200 unrelated families (173 from Czech Republic and 27 from Slovakia). More than 80% of all mutant alleles were identified, using a combination of PCR/RFLP, DGGE, TTGE, DHPLC, and sequencing. A total of 40 different mutations and 18 polymorphisms were detected on 400 independent mutant chromosomes. The most common molecular defect was H1069Q (57% of all 400 studied alleles). Each of the other 39 mutations was present in no more than 4% of WD alleles and 23 mutations were found in only one WD allele each (0.25%). Thirteen novel mutations were identified, including seven missense mutations (L641S, T737R, D918E, T1033S, G1111D, D1271N, and G1355C), four small deletions (19_20delCA, 1518_1522del5, 3140delA, and 3794_3803del10), and two splice-site mutations (2446-2A>G, 2865+1G>A). We did not find a significant correlation between H1069Q homozygosity and age of onset, and clinical and biochemical manifestation. Our data provide evidence that the H1069Q mutation-the most common molecular defect of the ATP7B gene in the Caucasian population-originates from Central/Eastern Europe. Screening of five prevalent mutations is predicted to reveal 70% of all mutant alleles presented in WD patients. This will provide a good starting point for early clinical classification of WD in our population.