Molecular Pathology Detection Strategies for Three Autosomal Dominant Neurodegenerative Diseases

The present study aimed to optimise mutation detection strategies for three autosomal dominant neurological diseases, myotonic dystrophy (DM), amyotrophic lateral sclerosis (ALS) and tuberous sclerosis complex (TSC). (A) Myotonic dystrophy: After exploring different methods that have been used for the detection of CTG repeat expansions in DM patients, a strategy was chosen, optimised and applied to screen 49 DM families (86 DM affected and 96 apparently normal individuals). Using published primer sets, both radiolabelled and non-radiolabelled PCR amplification of the area containing the CTG repeat were optimised using the published conditions as a starting point. After PCR optimisation, DM alleles carrying up to 90 CTG repeats were properly amplified however, amplification of > 90 CTG repeats was not possible due to PCR limitations. To detect such expansions, Southern blot analysis using the BglI enzyme and the p5B1.4 DNA probe was optimised. The running time and the voltage used for gel electrophoresis were modified to get clear separation of the second band that represents the expanded CTG repeats. Meanwhile, normal individuals showed only a single band so that they were not confused with the affected persons. Using this strategy, all CTG repeat expansions between 50 and several thousands were detected. Analysis of the results obtained revealed the following: 1) A correlation between the intergenerational repeat expansion and the patient phenotype giving legitimacy to the phenomenon of anticipation. 2) Two cases of reduction of the repeat size upon paternal transmission. Since the age of both asymptomatic daughters were younger than the age at onset of the disease in their fathers, it was not possible to anticipate their clinical outcome. 3) Lastly, a severely affected child with mental retardation and onset in infancy was found to be paternally transmitted. (B) Amyotrophic lateral sclerosis: Two mutations in the superoxide dismutase (SOD-1) gene, Ala4Val and Ilell3Thr, were previously shown to be prevalent among familial amyotrophic lateral sclerosis (FALS) patients. These two mutations changed restriction enzyme recognition sites, so that restriction digestion of the appropriate PCR products was optimised and used to screen for their presence in the studied amyotrophic lateral sclerosis patients. SSCP analysis was also optimised and applied as a screening method for detection of unknown mutations in the SOD-1 gene using DNA samples from 2 familial and 67 sporadic amyotrophic lateral sclerosis (ALS) patients. Any experiment with a positive SSCP screening result was repeated and a few false positive results due to PCR errors and/or errors in the gel preparations, loading or electrophoresis were detected. A reproducible SSCP band shift was detected in exon 4 from one of the two familial cases. Sequencing of that exon revealed a G277?C point mutation which caused a Gly93Arg missense change. This mutation was confirmed to be present in all affected members of that family. Gly93 is a neutral and polar amino acid and is highly conserved among 18 different species and it was substituted by the basic arginine. Mapping of Gly93 to the crystallographic structure of the SOD-1 gene revealed that it is one of the critical glycine residues that allow main chain conformation and packing interactions so that a mutation affecting this residue should have a deleterious effect on the conformation and stability of the enzyme dimer. In this FALS family, the affected members showed an early age of onset of the disease (26-40 years). Analysis of the results obtained revealed that: 1) In the two FALS families screened one SOD-1 mutation was detected in the two screened FALS patients. 2) No SOD-1 mutations could be detected in any of the sporadic cases. These results suggested that other gene(s) may be involved in the familial form of the disease and make it unlikely that SOD-1 mutations are major determinants of sporadic ALS. (C) Tuberous sclerosis: In order to screen the tuberous sclerosis complex (TSC) patients for point mutations within the TSC2 gene, chemical cleavage of the mismatch (CCM) analysis was optimised and applied to four RT-PCR amplified (from 22 patients) and two DNA PCR amplified (from 32 patients for one segment and from 10 patients for the other segment) TSC2 segments. These segments were chosen as they include proposed important functional parts of the gene. Using this approach, nine cleavage products were detected from the screened patients. Sequence analysis of the corresponding cDNA and/or DNA segments revealed three missense mutations in three sporadic TSC patients and three polymorphic changes. No mutations could be detected in the screened promoter area of the gene. (Abstract shortened by ProQuest.)

Erratum: The genomic architecture of NLRP7 is Alu rich and predisposes to disease-associated large deletions

NLRP7 is a major gene responsible for recurrent hydatidiform moles. Here, we report 11 novel NLRP7 protein truncating variants, of which five deletions of more than 1-kb. We analyzed the transcriptional consequences of four variants. We demonstrate that one large homozygous deletion removes NLRP7 transcription start site and results in the complete absence of its transcripts in a patient in good health besides her reproductive problem. This observation strengthens existing data on the requirement of NLRP7 only for female reproduction. We show that two other variants affecting the splice acceptor of exon 6 lead to its in-frame skipping while another variant affecting the splice donor site of exon 9 leads to an in-frame insertion of 54 amino acids. Our characterization of the deletion breakpoints demonstrated that most of the breakpoints occurred within Alu repeats and the deletions were most likely mediated by microhomology events. Our data define a hotspot of Alu instability and deletions in intron 5 with six different breakpoints and rearrangements. Analysis of NLRP7 genomic sequences for repetitive elements demonstrated that Alu repeats represent 48% of its intronic sequences and these repeats seem to have been inserted into the common NLRP2/7 primate ancestor before its duplication into two genes

Is There A Role For Oral L-Carnitine Therapy In Anemia And Cardiac Dysfunction Management In Egyptian Patients On Maintenance Hemodialysis?

Aim: L-carnitine is a short organic hydrosoluble molecule and is present in biological materials like free carnitine and acylcarnitines, which constitute the carnitine system. Long-term intermittent hemodialysis is associated with a reduction in plasma and tissue L-carnitine levels. Available studies on carnitine supplementation suggest the use of this molecule in dialysis, especially for those patients with cardiac complications, impaired exercise and functional capacities, and increased episodes of hypotension. Moreover, in some patients, the improved stability of erythrocyte membranes with L-carnitine supplementation may decrease erythropoietin requirements, thus leading to a reduction of dialytic costs. To study if there a possible advantageous effects for L– carnitine oral supplementation in anemia and cardiac dysfunction management in a cohort of Egyptian patients on maintenance hemodialysis. Methods: Fifty-five patients with chronic renal failure on maintenance hemodialysis were classified into 2 groups: L-carnitine group: 20 patients (12 male and 8 female, mean age 47.66±17.73 years, hemodialysis duration 51.36±18.14 months, subjected to three sessions/week reaching a Kt/V of 1.49±0.37) they received oral L-carnitine therapy 1.500 mg/day and control group: 35 patients (24 male and 11 female, mean age 37.9±14.7 years, hemodialysis duration 53.83±15.17 months, subjected to three sessions/week reaching Kt/V of 1.33±0.23). Both groups were on Erythropoietin therapy and IV iron whenever indicated. Echogardiographic studies were performed before and at the end of the study. Results: Serum hemoglobin were comparable in the L-carnitine group and control group at the start and six months after therapy (8.63±1.77 and 9.39±2.02 gm/dl, p= 0.18, 10.49±1.65 and 10.92±2.48 gm/dL p= 0.76 respectively). The weekly maintenance dose of Erythropoietin in spite of being lower in L-carnitine group (80.16±35.61 units/kg) compared to control group (91.9±38.21 units/kg) it does not reach a statistical significance (p= 0.20). No significant improvement could be observed in echogardiographic findings in the L-carnitine group after therapy. Conclusion: The role of L-carnitine in hemodialysis patients is questionable. Our study revealed no observed significant improvement in echocardiographic findings 6 months after therapy. However, -a statistically non significant-reduction in Erythropoietin dose was achieved in the L-carnitine-treated compared to the control group while maintaining comparable target hemoglobin in both groups. Long-term studies including larger number of patients are required to clarify its role in hemodialysis patients

Mutation spectrum analysis of Duchenne/Becker muscular dystrophy in 68 families in Kuwait: The era of personalized medicine

<div><p>Duchenne and Becker muscular dystrophies (DMD/BMD) are X-linked recessive neuromuscular disorders characterized by progressive irreversible muscle weakness and atrophy that affect both skeletal and cardiac muscles. DMD/BMD is caused by mutations in the <i>Dystrophin</i> gene on the X chromosome, leading to the absence of the essential muscle protein Dystrophin in DMD. In BMD, Dystrophin is partially functioning with a shorter protein product. Recent advances in molecular therapies for DMD require precise genetic diagnoses because most therapeutic strategies are mutation-specific. Hence, early diagnosis is crucial to allow appropriate planning for patient care and treatment. In this study, data from DMD/BMD patients who attended the Kuwait Medical Genetic Center during the last 20 years was retrieved from a Kuwait neuromuscular registry and analyzed. We combined multiplex PCR and multiplex ligation-dependent probe amplification (MLPA) with Sanger sequencing to detect <i>Dystrophin</i> gene mutations. A total of 35 different large rearrangements, 2 deletion-insertions (Indels) and 4 substitution mutations were identified in the 68 unrelated families. The deletion and duplication rates were 66.2% and 4.4%, respectively. The analyzed data from our registry revealed that 11 (16%) of the DMD families will benefit from newly introduced therapies (Ataluren and exon 51 skipping). At the time of submitting this paper, two cases have already enrolled in Ataluren (Tranlsarna™) therapy, and one case has been enrolled in exon 51 skipping therapy.</p></div

Graphical representation showing the extent of deletions/duplications in the <i>Dystrophin</i> gene in DMD and BMD patients.

<p>Exon numbers for the <i>Dystrophin</i> gene are indicated at the top. The number of cases having similar deletions/duplications is indicated in parenthesis. Deletions and duplications in the <i>Dystrophin</i> gene in DMD/BMD patients showing two major hot spots.</p

Mutation types in DMD/BMD patients.

<p>Mutation types in DMD/BMD patients.</p

Substitution and deletion-insertion (Indels) mutations in DMD patients.

<p>Substitution and deletion-insertion (Indels) mutations in DMD patients.</p

Distribution of DMD/BMD patients according to their nationality.

<p>Distribution of DMD/BMD patients according to their nationality.</p

The genomic architecture of NLRP7 is Alu rich and predisposes to disease-associated large deletions

WOS: 000384088000010PubMed ID: 26956250NLRP7 is a major gene responsible for recurrent hydatidiform moles. Here, we report 11 novel NLRP7 protein truncating variants, of which five deletions of more than 1-kb. We analyzed the transcriptional consequences of four variants. We demonstrate that one large homozygous deletion removes NLRP7 transcription start site and results in the complete absence of its transcripts in a patient in good health besides her reproductive problem. This observation strengthens existing data on the requirement of NLRP7 only for female reproduction. We show that two other variants affecting the splice acceptor of exon 6 lead to its in-frame skipping while another variant affecting the splice donor site of exon 9 leads to an in-frame insertion of 54 amino acids. Our characterization of the deletion breakpoints demonstrated that most of the breakpoints occurred within Alu repeats and the deletions were most likely mediated by microhomology events. Our data define a hotspot of Alu instability and deletions in intron 5 with six different breakpoints and rearrangements. Analysis of NLRP7 genomic sequences for repetitive elements demonstrated that Alu repeats represent 48% of its intronic sequences and these repeats seem to have been inserted into the common NLRP2/7 primate ancestor before its duplication into two genes.Max E. Binz Fellowship from the McGill University Faculty of Medicine; Canadian Institute of Health ResearchCanadian Institutes of Health Research (CIHR) [MOP-86546, PPP-122897]We thank the patients and their family for their cooperation. The authors wish to acknowledge the use of the Sequencing platform of the McGill University and Genome Quebec Innovation Centre. NMPN was supported by a Max E. Binz Fellowship from the McGill University Faculty of Medicine. The study was supported by the Canadian Institute of Health Research grants, MOP-86546 and PPP-122897, to RS