Identification of three novel alleles of HLA-DRB1 and HLA-A in the Brazilian population.

Two human leukocyte antigen (HLA)-DRB1 (HLA-DRB1*1376 and -DRB1*1465) and one HLA-A (HLA-A*2471) novel alleles have been identified in individuals from the Brazilian Bone Marrow Donor Registry. DNA sequencing of exon 2 for HLA-DRB1 alleles showed two and five nucleotide substitutions in -DRB1*1376 and -DRB1*1465, compared with closely related alleles, respectively. These substitutions result in a change of amino acid residues in HLA-DRB1*1376 at position 74 (Arg --> Glu) and in -DRB*1465 at positions 47 (Tyr --> Phe), 57 (Asp --> Ser) and 74 (Glu --> Ala). On the other hand, sequence analysis of exons 2 and 3 for HLA-A*2471 showed a single substitution, leading to a single amino acid change at position 151 (His --> Arg). These three novel alleles may have originated from other HLA alleles by gene conversion. However, it is also possible that HLA-A*2471 has evolved from one of the alleles of the HLA-A*2402 group through a point mutation.

Geographic distribution of hepatitis C virus genotypes in Brazil.

Brazil is a country of continental dimension with a population of different ethnic backgrounds. Thus, a wide variation in the frequencies of hepatitis C virus (HCV) genotypes is expected to occur. To address this point, 1,688 sequential samples from chronic HCV patients were analyzed. HCV-RNA was amplified by the RT-PCR from blood samples collected from 1995 to 2000 at different laboratories located in different cities from all Brazilian States. Samples were collected in tubes containing a gel separator, centrifuged in the site of collection and sent by express mail in a refrigerated container to Laboratório Bioquímico Jardim Paulista, São Paulo, SP, Brazil. HCV-RNA was extracted from serum and submitted to RT and nested PCR using standard procedures. Nested PCR products were submitted to cycle sequencing reactions without prior purification. Sequences were analyzed for genotype determination and the following frequencies were found: 64.9% (1,095) for genotype 1, 4.6% (78) for genotype 2, 30.2% (510) for genotype 3, 0.2% (3) for genotype 4, and 0.1% (2) for genotype 5. The frequencies of HCV genotypes were statistically different among Brazilian regions (P = 0.00017). In all regions, genotype 1 was the most frequent (51.7 to 74.1%), reaching the highest value in the North; genotype 2 was more prevalent in the Center-West region (11.4%), especially in Mato Grosso State (25.8%), while genotype 3 was more common in the South (43.2%). Genotypes 4 and 5 were rarely found and only in the Southeast, in São Paulo State. The present data indicate the need for careful epidemiological surveys throughout Brazil since knowing the frequency and distribution of the genotypes would provide key information for understanding the spread of HCV.

Geographic distribution of hepatitis C virus genotypes in Brazil

Brazil is a country of continental dimension with a population of different ethnic backgrounds. Thus, a wide variation in the frequencies of hepatitis C virus (HCV) genotypes is expected to occur. To address this point, 1,688 sequential samples from chronic HCV patients were analyzed. HCV-RNA was amplified by the RT-PCR from blood samples collected from 1995 to 2000 at different laboratories located in different cities from all Brazilian States. Samples were collected in tubes containing a gel separator, centrifuged in the site of collection and sent by express mail in a refrigerated container to Laboratório Bioquímico Jardim Paulista, São Paulo, SP, Brazil. HCV- RNA was extracted from serum and submitted to RT and nested PCR using standard procedures. Nested PCR products were submitted to cycle sequencing reactions without prior purification. Sequences were analyzed for genotype determination and the following frequencies were found: 64.9 percent (1,095) for genotype 1, 4.6 percent (78) for genotype 2, 30.2 percent (510) for genotype 3, 0.2 percent (3) for genotype 4, and 0.1 percent (2) for genotype 5. The frequencies of HCV genotypes were statistically different among Brazilian regions (P = 0.00017). In all regions, genotype 1 was the most frequent (51.7 to 74.1 percent), reaching the highest value in the North; genotype 2 was more prevalent in the Center-West region (11.4 percent), especially in Mato Grosso State (25.8 percent), while genotype 3 was more common in the South (43.2 percent). Genotypes 4 and 5 were rarely found and only in the Southeast, in São Paulo State. The present data indicate the need for careful epidemiological surveys throughout Brazil since knowing the frequency and distribution of the genotypes would provide key information for understanding the spread of HCV.

Absence of correlation between skewed X inactivation in blood and serum creatine-kinase levels in Duchenne/Becker female carriers.

The pattern of X inactivation in lymphocyte DNA was investigated in 107 Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) carriers (102 asymptomatic and 5 manifesting carriers) and 117 normal female controls of different ages, with the aim: a) to analyze the pattern of X inactivation in blood DNA of a large number of DMD/BMD carriers as compared to normal female controls; b) to determine if there is a decrease in serum creatine kinase (CK) levels with age in obligate DMD/BMD carriers; c) to determine if there is a correlation between X-chromosome inactivation and serum CK among asymptomatic DMD/BMD carriers of different ages or with different clinical manifestations in symptomatic carriers. A high proportion of females showed extremely skewed X inactivation (>90% of one X preferentially inactivated), which was almost the same among carriers and normal controls (19 and 24%, respectively). The mean serum CK was significantly greater among young (<20 years old) than adult (>20 years old) DMD/BMD carriers and it decreased significantly until age 20 with an apparent stabilization afterwards. No statistically significant correlation was found between the proportion of active X(DMD) in blood and serum CK activity in DMD/BMD carriers although it was higher among those less than 20 years old. Our observations suggest that highly skewed X-chromosome pattern in blood (with preferential inactivation of the X(N) chromosome) is not enough to predict that a young DMD carrier will develop muscular weakness.

Paternal inheritance or different mutations in maternally related patients occur in about 3% of Duchenne familial cases.

Duchenne dystrophy (DMD) is an X-linked lethal condition which affects 1 in 3,500 boys. The DMD gene is deleted in about 60-65% of patients while in the remaining 35-40% the condition is caused by point mutations, small insertions, or duplications. We have ascertained 967 DMD families (680 isolated and 287 familial cases). Screening for deletions showed a molecular deletion in 383 among 615 (62.3%) analyzed cases. However, 10 families were unusual: In 7 of them, 2 or more DMD patients were related through paternal lines while in 3 others, affected boys related through maternal lines carried different mutations or originated through independent new mutation events. The finding of 10 atypical genealogies, which represent about 1% of the sample (10/967) or about 3% of familial cases (10/287) is higher than we would expect by chance. Even so, it is an underestimate because screening of mutations in all the affected DMD relatives from each genealogy is not done in many of the familial cases. It suggests that other mechanisms (such as transposon-like elements, for example) could be responsible for a higher genomic instability leading to novel mutations as reported previously by us and others in DMD and in other genetic disorders such as hemophilia and inherited peripheral neuropathies. On the other hand, it shows the importance of testing all affected patients within each genealogy to prevent possible mistakes in carrier detection, genetic counseling, and prenatal diagnosis.

Novel point mutations in the dystrophin gene.

Duchenne (DMD) and Becker (BMD) type muscular dystrophies are allelic X-linked recessive disorders caused by mutations in the gene encoding dystrophin. About 65% of the cases are caused by deletions, while 5-10% are duplications. The remaining 30% of affected individuals may have smaller mutations (point mutations or small deletions/insertions) which cannot be identified by current diagnostic screening strategies. In order to look for pathogenic small mutations in the dystrophin gene, we have screened the 18 exons located in the hot spot region of this gene through two different single strand conformation polymorphism (SSCP) conditions. Five different pathogenic mutations were identified in 6 out of 192 DMD/BMD patients without detectable deletions: 2 nonsense, 1 bp insertion, 1 bp deletion and 1 intronic. Except for the intronic change, which alters a splice site, all the others cause a premature stop codon. In addition, 8 apparently neutral changes were identified. However, interestingly, one of them was not identified in 195 normal chromosomes, although it was previously described in a DMD patient from a different population. The possibility that this mutation may be pathogenic is discussed. Except for two neutral changes, all the others are apparently here described for the first time.

High proportion of new mutations and possible anticipation in Brazilian facioscapulohumeral muscular dystrophy families.

A gene responsible for facioscapulohumeral muscular dystrophy (FSHD) has been localized at 4q35. Subsequently, it was found that probe p13E-11 detects a polymorphic EcoRI fragment, usually > 28 kb, in normal individuals, whereas in sporadic and familial FSHD cases, an EcoRI fragment, usually < 28 kb, was found. Although these findings have been amply confirmed, several aspects are as yet either controversial or unsolved. In the present investigation, 34 Brazilian FSHD families were studied at the clinical and the molecular level for the following purposes: to assess the frequency of new mutations and their effect on estimates of biological fitness, to characterize FSHD-associated EcoRI fragments detected with probe p13E-11 in familial--as compared with isolated--FSHD cases, and to assess whether anticipation occurs in multigenerational families. Results from our study suggest that new mutations are apparently frequent for FSHD and may account for at least one-third of the cases, that somatic mosaicism may not be rare, and that biological fitness appeared to be reduced in FSHD, ranging from 0.6 to 0.82 by different estimates, with no difference in sexes. Interestingly, the size of the new EcoRI fragment is apparently smaller in more severely affected isolated patients. Moreover, the age at onset of clinical signs, as well as the age at ascertainment, in patients from multigenerational families suggests that anticipation occurs for FSHD in the majority of the families.

A Caucasian family with the 3271 mutation in mitochondrial DNA.

The second most common mutation associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) in Japan is the 3271 mutation. This mutation was found in a Brazilian family of Portuguese and Italian descent, indicating that this mutation also exists in a race other than Japanese. The propositus had mild clinical manifestations atypical of MELAS, suggesting that patients with the 3271 mutation exhibit heterogeneous phenotypic expression as seen in the 3243 mutation.

A deletion including the brain promoter of the Duchenne muscular dystrophy gene is not associated with mental retardation.

A total of 161 unrelated Duchenne (DMD) and Becker muscular dystrophy (BMD) patients were screened for deletions in the brain promoter region of the dystrophin gene. Southern blot analysis using a probe for the brain promoter detected a deletion in this region in only one of the DMD families, in a patient with normal intelligence. This deletion also included the promoter of the muscle-type dystrophin and the exons encoding the actin-binding and part of the spectrin-like domains. Our data suggest that deletions in the brain promoter region are rare in DMD and are compatible with normal intelligence.