NHEJ-Mediated Repair of CRISPR-Cas9-Induced DNA Breaks Efficiently Corrects Mutations in HSPCs from Patients with Fanconi Anemia.

Non-homologous end-joining (NHEJ) is the preferred mechanism used by hematopoietic stem cells (HSCs) to repair double-stranded DNA breaks and is particularly increased in cells deficient in the Fanconi anemia (FA) pathway. Here, we show feasible correction of compromised functional phenotypes in hematopoietic cells from multiple FA complementation groups, including FA-A, FA-C, FA-D1, and FA-D2. NHEJ-mediated repair of targeted CRISPR-Cas9-induced DNA breaks generated compensatory insertions and deletions that restore the coding frame of the mutated gene. NHEJ-mediated editing efficacy was initially verified in FA lymphoblastic cell lines and then in primary FA patient-derived CD34+ cells, which showed marked proliferative advantage and phenotypic correction both in vitro and after transplantation. Importantly, and in contrast to homologous directed repair, NHEJ efficiently targeted primitive human HSCs, indicating that NHEJ editing approaches may constitute a sound alternative for editing self-renewing human HSCs and consequently for treatment of FA and other monogenic diseases affecting the hematopoietic system.

Utility of two SMN1 variants to improve spinal muscular atrophy carrier diagnosis and genetic counselling.

Spinal muscular atrophy (SMA) is caused by deletions/mutations in SMN1. Most heterozygous SMA carriers have only one SMN1 copy in one of the alleles (1/0 carriers). However, a few carriers lack SMN1 in one of their chromosomes, but present two gene copies in the other. These "2/0 carriers" are undistinguishable from non-carrier individuals (1/1) with currently available methods. Previous association of SMN1 variants c.*3 + 80 T > G and c.*211_*212del with two SMN1 copies in cis in Ashkenazi population prompted us to analyze them in 270 Spanish individuals (SMA carriers, patients and general population). Both variants were much more frequently detected in chromosomes with 2 SMN1 copies in cis in comparison with chromosomes carrying one copy (17.9 vs. 0.7%; p < 0.001). In particular, one-fifth of 2/0 SMA carriers harboured one or both variants compared to none of 99 non-carriers with two SMN1 copies (p < 0.001). The c.*211_*212del variant was also much more frequent in exon 8 of SMN2-SMN1 hybrids than in that of intact SMN1 genes (20 vs. 0.83%, p < 0.001), suggesting its association with chromosomal rearrangements. Although absence of these variants does not exclude that a particular individual is a 2/0 SMA carrier, their presence is valuable to substantially increase residual risk in putative carriers, thus improving genetic counselling.

Correlation between SMA type and SMN2 copy number revisited: An analysis of 625 unrelated Spanish patients and a compilation of 2834 reported cases.

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by loss or mutations in SMN1. According to age of onset, achieved motor abilities, and life span, SMA patients are classified into type I (never sit), II (never walk unaided) or III (achieve independent walking abilities). SMN2, the highly homologous copy of SMN1, is considered the most important phenotypic modifier of the disease. Determination of SMN2 copy number is essential to establish careful genotype-phenotype correlations, predict disease evolution, and to stratify patients for clinical trials. We have determined SMN2 copy numbers in 625 unrelated Spanish SMA patients with loss or mutation of both copies of SMN1 and a clear assignation of the SMA type by clinical criteria. Furthermore, we compiled data from relevant worldwide reports that link SMN2 copy number with SMA severity published from 1999 to date (2834 patients with different ethnic and geographic backgrounds). Altogether, we have assembled a database with a total of 3459 patients to delineate more universal prognostic rules regarding the influence of SMN2 copy number on SMA phenotype. This issue is crucial in the present scenario of therapeutic advances with the perspective of SMA neonatal screening and early diagnosis to initiate treatments.

Inducible and Acquired Clarithromycin Resistance in the Mycobacterium abscessus Complex.

PURPOSE: Clarithromycin was considered the cornerstone for the treatment of Mycobacterium abscessus complex infections. Genetic resistance mechanisms have been described and many experts propose amikacin as an alternative. Nevertheless, clarithromycin has several advantages; therefore, it is necessary to identify the non-functional erm(41) allele to determine the most suitable treatment. The aims of this study were to characterize the molecular mechanisms of clarithromycin resistance in a collection of Mycobacterium abscessus complex isolates and to verify the relationship between these mechanisms and the antibiogram. MATERIALS AND METHODS: Clinical isolates of M. abscessus complex (n = 22) from 16 patients were identified using four housekeeping genes (rpoB, secA1, sodA and hsp65), and their genetic resistance was characterized by studying erm(41) and rrl genes. Nine strains were recovered from the clinical isolates and subjected to E-test and microdilution clarithromycin susceptibility tests, with readings at 3, 7 and 14 days. RESULTS: We classified 11/16 (68.8%) M. abscessus subsp. abscessus, 4/16 (25.0%) M. abscessus subsp. bolletii, and 1/16 (6.3%) M. abscessus subsp. massiliense. T28 erm(41) allele was observed in 8 Mycobacterium abscessus subps. abscessus and 3 Mycobacterium abscessus subsp. bolletii. One strain of M. abscessus subsp. bolletii had an erm(41) gene truncated and was susceptible to clarithromycin. No mutations were observed in rrl gene first isolates. In three patients, follow-up of initial rrl wild-type strains showed acquired resistance. CONCLUSIONS: Most clinical isolates of M. abscessus complex had inducible resistance to clarithromycin and total absence of constitutive resistance. Our findings showed that the acquisition of resistance mutations in rrl gene was associated with functional and non-functional erm(41) gene. Caution is needed when using erm(41) sequencing alone to identify M. abscessus subspecies. This study reports an acquired mutation at position 2057 of rrl gene, conferring medium-low clarithromycin constitutive resistance.

Fungemia in a Spanish hospital: the role of Candida parapsilosis over a 15-year period.

BACKGROUND: Candida parapsilosis is one of the main causes of fungemia in tertiary-care hospitals. Few studies have analysed the changes in its distribution over a long period. We compared the distribution of C. parapsilosis with that of other fungi over a 15-y period in a tertiary hospital. METHODS: The susceptibility of C. parapsilosis was analysed using the new species-specific clinical breakpoints. The C. parapsilosis complex species were differentiated molecularly. RESULTS: From January 1997 to December 2011, 360 isolates causing 350 episodes of fungemia were isolated. C. parapsilosis was the second most frequently isolated species (20%); only 1 C. orthopsilosis was identified and there were no C. metapsilosis. The remaining episodes were caused by C. albicans (43.1%), C. tropicalis (14.4%), C. glabrata (11.7%), and other fungal species (10.8%). The incidence of candidemia increased more than two-fold between 2009 and 2011 (from 3.3 to 7.4 cases/100,000 population), and C. parapsilosis and C. glabrata fungemia increased throughout the period. C. parapsilosis was the most frequent species in children under 15 y (57.1%). All C. parapsilosis isolates were susceptible to anidulafungin, micafungin, flucytosine, amphotericin B, and posaconazole, while 98.5% were susceptible to caspofungin, 97.1% to voriconazole, 95.6% to fluconazole, and 76.5% to itraconazole. CONCLUSIONS: This long-term study showed a slight increase in the incidence of candidemia during the years of the study and a trend towards an increase in C. parapsilosis. Because of its high frequency and intrinsic low susceptibility to echinocandins, the prevalence and susceptibility of C. parapsilosis should be monitored, especially in children.

Factors associated with differences between conventional contact tracing and molecular epidemiology in study of tuberculosis transmission and analysis in the city of Barcelona, Spain.

The aim of this study was to analyze the factors associated with conventional contact tracing (CCT) and molecular epidemiology (ME) methods in assessing tuberculosis (TB) transmission, comparing the populations studied and the epidemiological links established by both methods. Data were obtained from TB case and CCT registries, and ME was performed using IS6110-based restriction fragment length polymorphism (RFLP) analysis and mycobacterial interspersed repetitive unit 12 (MIRU12) typing as a secondary typing method. During two years (2003 and 2004), 892 cases of TB were reported, of which 687 (77%) were confirmed by culture. RFLP analysis was performed with 463 (67.4%) of the 687 isolated strains, and MIRU12 types in 75 strains were evaluated; 280 strains (60.5%) had a unique RFLP pattern, and 183 (39.5%) shared patterns, grouping into 65 clusters. CCT of 613 (68.7%) of 892 cases detected 44 clusters involving 101 patients. The results of both CCT and ME methods yielded 96 clusters involving 255 patients. The household link was the one most frequently identified by CCT (corresponding to 80.7% of the cases clustered by this method), whereas nonhousehold and unknown links were associated with 94.1% of the strains clustered by ME. When both methods were used in 351 cases (39.3%), they showed the same results in 214 cases (61%). Of the remainder, 106 (30.2%) were clustered only by ME, 19 (5.5%) were clustered only by CCT, and 12 (3.4%) were clustered by both methods but into different clusters. Patients with factors potentially associated with social problems were less frequently studied by CCT (P = 0.002), whereas patients of <15 years of age, most with negative cultures, were less frequently studied by ME (P = 0.005). Significant differences in the populations studied by ME versus CCT were observed, possibly explaining the scarce correlation found between the results of these methods. Moreover, ME allowed the detection of nonhousehold contact relationships, whereas CCT was more useful for tracing transmission chains involving patients of <15 years of age. In conclusion, the two methods are complementary, suggesting the need to improve the methodology of contact study protocols.

Group A streptococcal infections in injection drug users in Barcelona, Spain: epidemiologic, clinical, and microbiologic analysis of 3 clusters of cases from 2000 to 2003.

An unexplained resurgence of Group A streptococci (GAS) infections has been observed since the mid-1980s in the United States and Europe, particularly among intravenous drug users (IDUs). Several risk factors have been identified. Mutations in the capsule synthesis regulator genes (csrRS) have been associated with an increase in virulence. From January 1998 to December 2003, we conducted a prospective and retrospective descriptive analysis of invasive GAS soft-tissue infections in IDUs in Barcelona, Spain. Clinical features were collected, and we conducted a surveillance study to identify risk factors associated with GAS soft-tissue infections. We analyzed chromosomal DNA by low cleavage restriction enzymes and used pulsed-field gel electrophoresis (PFGE) and variable gene sequence typing (VGST) of the emm gene to disclose the epidemiologic relationship between the strains. We analyzed the influence of clonality (M-type) and mutations in csrRS genes of these strains on clinical features. We identified 44 cases, all of which were grouped in 3 clusters: fall 2000, fall 2002, and fall 2003. Cellulitis with or without abscesses (75%) and fever (90.9%) were the most common clinical manifestations. Distant septic complications were infrequent (18.2%). Although all patients had severe infections (mainly bacteremic needle abscesses), their outcome with antibiotic therapy, usually beta-lactam, was successful in all cases. However, surgery was needed in 40.9% of patients. Through the surveillance study we found that infected patients had a higher number of drug injections per day (odds ratio [OR], 18.84; 95% confidence interval [CI], 4.83-79.4; p<0.00001), shared paraphernalia for drug use more frequently (OR, 11.11; 95% CI, 3.24-39.04; p<0.0001), were in a higher proportion both currently unemployed and homeless (OR, 4.22; 95% CI, 1.5-12.15; p<0.0001), were not in a methadone maintenance program (OR, 0.03; 95% CI, 0-0.19; p<0.00001), and more often bought drugs at a specific site (OR, 33.92; 95% CI, 7.44-174.93; p<0.00001) and from a specific dealer (OR, 72; 95% CI, 8-3090; p<0.00001), compared with patients not infected. The fall 2000 cluster was polyclonal, whereas the other 2 clusters were mainly due to the same strain of GAS (emm 25.2), and were defined as epidemic outbreaks. Clinically, the cases due to the clonal strain presented abscesses and needed surgery more frequently (p<0.001 and p=0.005, respectively). On the other hand, mutations in the csrRS genes were not associated with invasive GAS soft-tissue infection. There has been an increase in the number of cases of invasive GAS soft-tissue infections in IDUs in Barcelona, which seems to be related to drug users' habits and their socioeconomic status. Clonality (emm 25.2) but not mutations in the csrRS genes was associated with more severe GAS soft-tissue infections.

Endemic meningococcal disease in Cerdanyola, Spain, 1987--93: molecular epidemiology of the isolates of Neisseria meningitidis.

OBJECTIVE: To establish the relationships between 30 Neisseria meningitidis strains isolated in Cerdanyola (Spain) from 30 out of 36 sporadic cases of meningococcal disease (MD) during 1987--93 and their spread in this population by multilocus enzyme electrophoresis (MEE) and by pulsed-field gel electrophoresis (PFGE), and to evaluate the usefulness of PFGE versus serologic typing methods and MEE as an alternative epidemiologic marker to study meningococcal infection. METHODS: Serotyping, electrophoretic mobility of seven isoenzymes determined by MEE and chromosomal DNA macrorestriction with NheI resolved by PFGE were analyzed. RESULTS: Of these 30 strains, 25 were serogroup B and the remaining five were serogroup C, with the 4:P1.15 and the 2b:NT as the most common antigenic phenotypes, respectively. There were 13 electrophoretic types (ETs) by MEE, with 14 isolates showing an identical ET, 8. Sixteen pulse types (PTs) were generated by PFGE. The 14 ET 8 isolates were clustered into six PTs, A1, A2, A4, A5, A6 and A8. However, by combining both methods, 19 genetically distinct groups were obtained. Eleven of these groups (20 serogroup B strains) and two of these (four serogroup C strains) were genetically related. CONCLUSIONS: We conclude that, according to the clonal population structure, these 30 N. meningitidis strains are heterogeneous although a great number are related. Moreover, PFGE is a useful method to establish clonal structure in N. meningitidis strains under endemic conditions. Finer discrimination of these strains was achieved by combining both MEE and PFGE methods.

Comparison of macrorestriction analysis of genomic DNA by pulsed-field gel electrophoresis and ribotyping with conventional methods for differentiation of Escherichia coli 0124 isolates.

OBJECTIVE: To evaluate the usefulness of different phenotypic and genotypic markers for epidemiological typing of enteroinvasive Escherichia coli 0124 (EIEC 0124). METHODS: Seven sporadic EIEC 0124 isolates and 22 isolates from two different outbreaks were characterized. Chromosomal deoxyribonucleic acid (DNA) macrorestriction analysis with XbaI resolved by pulsed-field gel electrophoresis (PFGE) and ribotyping with each of the three restriction endonucleases BglII EcoRI, and ClaI were compared with biotyping, antimicrobial susceptibility patterns and plasmid profiles. RESULTS: Biotypes and antimicrobial resistance profiles of the outbreak-associated strains showed considerable variation, thereby limiting the usefulness of such phenotypic markers. Only 57% of the sporadic isolates harbored plasmids. Three different ribotypes based on 5 to 7 bands were recorded among sporadic isolates whereas all outbreak-associated strains showed the same ribotype. BglII appeared to give the best discrimination whereas EcoRI and ClaI provided no additional information. Sporadic EIEC 0124 isolates showed a marked diversity of macrorestriction patterns (similarity coefficient 58 to 93%) and five different patterns were detected. In contrast, the outbreak isolates were closely related (similarity coefficient 90 to 100%). Genomic DNA macrorestriction analysis correlated well with ribotyping, but PFGE was more discriminating. CONCLUSIONS: PFGE is a useful method for epidemiological comparison and differentiation of EIEC 0124 isolates.