Inverse correlation between SMN1 and SMN2 copy numbers: evidence for gene conversion from SMN2 to SMN1

Most carriers of autosomal recessive spinal muscular atrophy (SMA) have only one copy of SMN1 because of SMN1 gene deletions or gene conversions from SMN1 to SMN2, which has only one base difference in coding sequence from SMN1. Using SMN gene dosage analysis, we determined the copy numbers of SMN1 and SMN2 in the general population as well as in SMA patients and carriers. Increased SMN1 copy number is associated with decreased SMN2 copy number in the general population; that is, SMN2 copy number was decreased to one or zero copies in 11 of 13 individuals with three or four copies of SMN1, whereas only 71 of 164 individuals with two copies of SMN1 had one or zero copies of SMN2 (P<0.01). SMN2 copy number was increased to three or four in a subset of SMN1 deletion/conversion carriers, and in most SMA patients with a milder phenotype. In conclusion, our data provide evidence that gene conversion from SMN2 to SMN1 occurs, and that SMN1 converted from SMN2 is present in the general population.     

Quantitative analysis of SMN1 gene and estimation of SMN1 deletion carrier frequency in Korean population based on real-time PCR

Spinal muscular atrophy (SMA) is an autosomal recessive disorder, caused by homozygous absence of the survival motor neuron gene (SMN1) in approximately 94% of patients. Since most carriers have only one SMN1 gene copy, several SMN1 quantitative analyses have been used for the SMA carrier detection. We developed a reliable quantitative real-time PCR with SYBR Green I dye and studied 13 patients with SMA and their 24 parents, as well as 326 healthy normal individuals. The copy number of the SMN1 gene was determined by the comparative threshold cycle (Ct) method and albumin was used as a reference gene. The homozygous SMN1 deletion ratio of patients was 0.00 and the hemizygous SMN1 deletion ratio of parents ranged from 0.39 to 0.59. The deltadelta Ct ratios of 7 persons among 326 normal individuals were within the carrier range, 0.41-0.57. According to these data, we estimated the carrier and disease prevalence of SMA at 1/47 and 1/8,496 in Korean population, respectively. These data indicated that there would be no much difference in disease prevalence of SMA compared with western countries. Since the prevalence of SMA is higher than other autosomal recessive disorders, the carrier detection method using real-time PCR could be a useful tool for genetic counseling.     

Determination of SMN1 and SMN2 copy number using TaqMan technology

Infantile spinal muscular atrophy (SMA) is a neuromuscular disease caused by homozygous deletion of the SMN1 gene in more than 90% of patients. Identification of carriers for the SMN1 deletion is important for diagnostic purposes and for genetic counseling. The current practical implications of SMN2 copy number determination are limited but may be important, for example, for future drug trials. Here we present a new rapid and reliable approach to determine the copy numbers of the SMN1 and SMN2 genes: For differentiation of the two genes, we developed a quantitative test on the basis of TaqMan technology using minor groove binder (MGB) probes. To evaluate the approach in respect to detection of SMN1 deletion carriers, we tested 40 putative carriers as well as 100 controls. We confirmed the carrier status in all individuals; furthermore, the distribution of SMN1 and SMN2 copies in the control cohort corresponded to that published previously. In total, a clear-cut differentiation between the different copy number ranges could be observed for both genes. This distinct differentiation is based on the exact specificity of MGB probes and the parallel analysis of an external reference locus that circumvents the problem of unavoidable deviations in DNA concentrations. The simplicity and reproducibility of the TaqMan assay presented here should facilitate its establishment in molecular diagnostic laboratories. Nevertheless, the applicability of quantitative analyses of SMN copy numbers requires knowledge about its options and limitations, based on the complex nature of the SMN region and the clinical variability of SMA. Therefore, determination of SMN1 and SMN2 copy numbers should only be offered after careful consideration in each case.     

Quantitative analysis of SMN1 and SMN2 genes based on DHPLC: a highly efficient and reliable carrier-screening test

Autosomal recessive spinal muscular atrophy (SMA) is a common, fatal neuromuscular disease caused by homozygous absence of the SMN1 gene in approximately 94% of patients. However, a highly homologous SMN2 gene exists in the same chromosome interval, centromeric to SMN1, and hampers detection of SMN1. We present a new, rapid, simple, and highly reliable method for detecting the SMN1 deletion/conversion and for determining the copy numbers of the SMN1 and SMN2 genes by DHPLC. We analyzed SMN1/SMN2 gene exon 7 deletion/conversion by DHPLC. A total of 25 patients with spinal muscular atrophy lacking the SMN1 gene as well as 309 control individuals from the general population and the family members of patients with SMA were analyzed. By DHPLC analysis, we could detect the SMA-affected cases efficiently just by recognizing an SMN2-only peak. Furthermore, after specific primer amplification and adjustment of the oven temperature, all of the SMA carriers with an SMN1/SMN2 ratio not equal to 1 could be identified unambiguously by this simple and efficient detection system. To calculate the total SMN1/SMN2 gene dosages further, we developed a specific multiplex competitive PCR protocol by simultaneously amplifying the CYBB gene (X-linked), the KRIT1 gene (on chromosome arm 7q), and the SMN1/SMN2 gene ratio by DHPLC. By applying this technique, we could successfully designate all of the genotypes with different SMN1/SMN2 gene copy numbers, including equal and unequal amounts of SMN1 and SMN2. We demonstrated that DHPLC is a fast and reliable tool for detection of carriers of SMA.     

Homozygous SMN1 deletions in unaffected family members and modification of the phenotype by SMN2

Spinal muscular atrophy is a common autosomal recessive neuromuscular disorder caused by the homozygous loss of the SMN1 gene. The absence of the SMN1 gene has been shown to occur in all types of SMA, childhood and adult forms. In rare cases, asymptomatic family members have also been found with homozygous mutations in the SMN1 gene, suggesting a role for phenotypic modifiers. We describe three unrelated asymptomatic individuals, with family histories of SMA, who were shown to have the homozygous SMN1 deletion. Quantitative studies indicated that the three individuals all had increased SMN2 copy numbers. These cases not only support the role of SMN2 in modifying the phenotype, but our data also demonstrate that expression levels consistent with five copies of the SMN2 genes maybe enough to compensate for the absence of the SMN1 gene. Lastly, in cases similar to the ones described, the measurement of the SMN2 gene copy number may provide valuable prognostic information.     

A novel DRB1*13 allele (DRB1*1327) on a DR17, DQ2 haplotype with a DRB1*0301 sequence motif in the 2nd hyperpolymorphic region

In a sample from a Netherlands Caucasian, we found a new DRB1*13 allele ( DRB1*1327 ). The nucleotide sequence of the second exon of the novel allele was identical to DRB1*1301 except for a single productive base substitution changing codon 26 from TTC to TAC, encoding phenylalanine and tyrosine, respectively. The new allele shares sequence with DRB1*03011 from codons 5 to 66. The haplotype carrying the new allele was, from known linkage disequilibria, deduced to be DRB1*1327, DRB3*0101, DQA1*05011, DQB1*0201 , i.e. similar to the DR17, DQ2 haplotype, which suggests that the DRB1*1327 allele has arisen by a double recombination event between a DR13 donor haplotype and a DR17, DQ2 recipient haplotype.     

Antigen, allele, and haplotype frequencies report of the ASHI minority antigens workshops: part 1, African-Americans

HLA typing was performed on 977 African Americans residing throughout most of the United States. Class I and class II antigens and class II alleles were defined for all individuals and class I alleles were determined for a subset of individuals. The occurrence of 854 of the individuals in family groups permitted direct counting of allele and haplotype frequencies. The data were analyzed for antigen, allele, and haplotype frequencies; recombination frequencies; segregation distortion; distribution of haplotype frequencies; linkage disequilibria; and geographic distribution of DR antigens. Tables of the antigen, allele, the most common two and three point haplotypes, and 88 extended haplotypes that include class I and class II alleles are presented. Notable findings include a lower than expected frequency of recombination between the B and DR loci (theta= 0.0013), lower than expected frequency of inheritance (44.5% vs 54.5%) of the DRB1*1503; DQB1*0602 haplotype, lower than anticipated linkage disequilibrium values for DR; DQ haplotypes, and a skewed geographic distribution of DR antigens.     

HLA-A, -B, -C, -DRB1 and -DQB1 allele and haplotype frequencies in the Serbian population

This study provides the first published detailed analysis of five loci polymorphisms as well as reports of two, three and five loci haplotype frequencies in the Serbian population in a sample of 1992 volunteer bone marrow donors recruited from different part of the country. Typing was performed by PCR SSO method combined with PCR SSP techniques to resolve ambiguities. In total, 16 HLA-A, 28 HLA-B, 14 HLA-C, 13 HLA-DRB1 and 5 HLA-DQB1 allelic groups were identified. The most frequent in allele groups are HLA-A^∗02 (29.5%), HLA-A^∗01 (14.2%), HLA-B^∗35 (13.1%), HLA-B^∗51 (12.8%), HLA-C^∗07 (24.8%), HLA-DRB1^∗11 (16.9%), HLA-DRB1^∗13 (13.2%), HLA-DQB1^∗03 (33.3%) and DQB1^∗05 (33.0%). The most frequent three- and five-loci haplotypes were A^∗01-B^∗08-DRB1^∗03 (5.9%) and A^∗02-B^∗18-DRB1^∗11 (1.9%), A^∗01-B^∗08-C^∗07-DRB1^∗03-DQB1^∗02 (6.6%) followed by A^∗02-B^∗18-C^∗07-DRB1^∗11-DQB1^∗03 (2.5%), then A^∗33-B^∗14-C^∗08-DRB1^∗01-DQB1^∗05 and A^∗02-B^∗35-C^∗04-DRB1^∗16-DQB1^∗05 (2.2% both), respectively. The results of cluster analysis showed that the Serbian population is closely related to the populations living in central Balkan and neighboring European regions. The level of allelic diversity found in this study are relevant to facilitate searching for unrelated matched donor and provide a healthy control population from our region that should be useful in the future disease association study.     

Survival motor neuron SMN1 and SMN2 gene promoters: identical sequences and differential expression in neurons and non-neuronal cells

Spinal muscular atrophy (SMA) is a recessive disorder involving the loss of motor neurons from the spinal cord. Homozygous absence of the survival of motor neuron 1 gene (SMN1) is the main cause of SMA, but disease severity depends primarily on the number of SMN2 gene copies. SMN protein levels are high in normal spinal cord and much lower in the spinal cord of SMA patients, suggesting neuron-specific regulation for this ubiquitously expressed gene. We isolated genomic DNA from individuals with SMN1 or SMN2 deletions and sequenced 4.6 kb of the 5' upstream regions of the these. We found that these upstream regions, one of which is telomeric and the other centromeric, were identical. We investigated the early regulation of SMN expression by transiently transfecting mouse embryonic spinal cord and fibroblast primary cultures with three transgenes containing 1.8, 3.2 and 4.6, respectively, of the SMN promoter driving beta-galactosidase gene expression. The 4.6 kb construct gave reporter gene expression levels five times higher in neurons than in fibroblasts, due to the combined effects of a general enhancer and a non-neuronal cell silencer. The differential expression observed in neurons and fibroblasts suggests that the SMN genes play a neuron-specific role during development. An understanding of the mechanisms regulating SMN promoter activity may provide new avenues for the treatment of SMA.     

Stability and haplotype analysis of the FRAXE region

FRAXE full mutations are rare and appear to be associated with mild mental retardation. As part of a screening survey of boys with learning difficulties to determine the frequency of full and premutations, we have collected data on the frequency of instability at FRAXE for about 4000 transmissions and the haplotype for over 7000 chromosomes. The distribution of FRAXE repeats was similar to other English populations but differed from two North American Caucasian series. Observed instability at FRAXE was rare but increased with increasing repeat number, and there were no expansions into the full mutation range, except in pedigrees ascertained through a full mutation. Haplotype analysis suggested division into five groups with each group having a characteristic distribution of FRAXE repeats. Fourteen of the 15 full mutations occurred on a single haplotype and this haplotype also had a significant excess of intermediate-sized alleles, suggesting that full mutations originate from large normal alleles. However, a related haplotype also had a significant excess of intermediates but we observed no full mutations on this haplotype, suggesting either loss or gain of stability determinants on it. We suggest that whilst triplet repeat size is a significant predisposing factor for expansion at FRAXE other genetic determinants are also likely to be important.     

The exon 2b region of the spinal muscular atrophy protein, SMN, is involved in self-association and SIP1 binding

Spinal muscular atrophy (SMA) is caused by mutations in the SMN (survival of motor neurons) gene and there is a correlation between disease severity and levels of functional SMN protein. Studies of structure-function relationships in SMN protein may lead to a better understanding of SMA pathogenesis. Self-association of the spinal muscular atrophy protein, SMN, is important for its function in RNA splicing. Biomolecular interaction analysis core analysis now shows that SMN self-association occurs via SMN regions encoded by exons 2b and 6, that exon 2b encodes a binding site for SMN-interacting protein-1 and that interaction occurs between exon 2- and 4-encoded regions within the SMN monomer. The presence of two separate self-association sites suggests a novel mechanism by which linear oligomers or closed rings might be formed from SMN monomers.     

HLA-A, -B, -C and -DRB1 allele and haplotype frequencies in the Macedonian population based on a family study

Aim

The aim of this study was to determine HLA allele and 2-, 3- and 4-loci haplotype frequencies in a sample from Macedonian population with defined haplotypes based on family history.

Refining the primary open-angle glaucoma GLC1C region on chromosome 3 by haplotype analysis

The GLC1C locus for primary open-angle glaucoma (POAG) is inherited as an autosomal dominant trait. This region on chromosome 3 is 11 cM long. DNA samples from members of a Greek and an American GLC1C family were obtained to determine whether additional typing of microsatellite markers in family members might narrow the region. GLC1C family members were evaluated clinically for POAG on the basis of open angles, intraocular pressures, cupping of discs, and visual fields. DNA samples from the Greek and Oregon GLC1C families were used to further refine the GLC1C region using microsatellite markers. A total of 22 affected members were identified in the two families. Common alleles for D3S3637 and D3S3612 were present in the disease haplotype from both families, suggesting that they may have a common founder. A newly diagnosed patient in the American family had a recombination in the distal portion of the GLC1C haplotype. This recombination narrows the GLC1C region from 11 to 4 cM.     

High frequency of the TCRBV20S1 null allele in the Sardinian population

Single nucleotide polymorphisms (SNPs) in the T-cell receptor (TCR) gene segments might play a role in shaping the TCR repertoire. Three polymorphisms have been described for the TCRBV20S1 gene segment, one of which is responsible for a nucleotide substitution at position 524, resulting in the introduction of a stop codon. Individuals homozygous for this inactivating polymorphism ("null allele") are unable to express TCRBV20 gene products. Using DNA restriction digestion analysis, we investigated the frequency of this polymorphism in 111 healthy Sardinian subjects. Inhabitants of the Mediterranean island of Sardinia are considered to represent a genetically isolated population. Our analyses revealed an incidence of 19.8% of homozygosity for the null allele, corresponding to an allele frequency of 0.45. Such an incidence, significantly higher than the one detected in 83 non-Sardinian Caucasians (6%), is the most elevated so far reported in the literature. BV20 is a single member subfamily and the null allele produces a gap in the potential TCR repertoire. Therefore, it is possible that an undetermined selective pressure could have played a role in determining the high frequency of this inactivating polymorphism in Sardinians. Alternatively, this finding could be related to a founder effect in this ancient island population.     

HIV-1 subtype frequency in Northeast Brazil: A systematic review and meta-analysis

Information on human immunodeficiency virus (HIV) molecular epidemiology is required to verify HIV/AIDS (acquired immune deficiency syndrome) epidemic dynamics in different regions, as well as provide support for response to antiretroviral therapy, transmission of resistance mutations, disease progression, and viral spread. The aim of this study was to conduct a systematic review and meta-analysis of the frequency of HIV-1 subtypes in Northeast Brazil. Seventy-six articles that refer to HIV-1 and its subtypes in the Northeast Brazil and published between 1 January 1999 and 31 August 2019 were identified. We included 27 articles for the qualitative synthesis, thus analyzing results from 4466 patients and 4298 genomic sequences. The results showed that subtypes B, F, and C and recombinant BF were responsible for 76% (IC95%: 71-80), 8% (IC95%: 5-11), 2% (IC95%: 2-3), and 7% (IC95%: 4-12) infections, respectively. The highest proportion of subtype B infections (82.2%) was observed in Piauí, while the subtype F had a high frequency in Pernambuco (23.4%). Bahia presented 11.6% of the proportion of recombinant BF. In addition, several recombinants such as AG, BC, BCF, and BD have been identified in the region. This is the first systematic review and meta-analysis on the HIV-1 subtype distribution in Northeast Brazil and has shown a high circulating viral diversity. Although subtype B is predominant in Brazil, a large frequency of non-B subtypes has also been found, which may have consequences for response to antiretroviral therapy, disease progression, and transmission. Thus, HIV molecular epidemiological data are essential for epidemic prevention and control strategies.     

广州4194份脐带血HLA-A、B、DRB1等位基因型和单倍型频率研究

目的 对广州脐血库10年来保存的脐血人类白细胞抗原(human leukocyte antigen,HLA)等位基因及单倍型分布特征进行分析.方法 采用单克隆板,序列特异引物聚合酶链反应,PCR序列特异性寡核苷酸探针和DNA测序分型方法对广州脐血库内4194份脐带血进行HIA-A、B、DRB1等位基因分型.用Arlequm软件计算HLA基因频率和单倍型频率.结果 在广州脐血库中,HLA-A、B、DRB1等位基因型分别有18,43,13种.累积频率>50%的显著高频率等位基因是:A*11,A*02,A*24,A*33,B*40,B*15,B*46,B*13,DRB1*12,DRB1*15,DRB1*09,DRB1*04;最常见的单倍型为:A2-B46、B6-DR9、A11-DR12、A2-B6-DR9.结论 广州脐血库脐血捐献者HIA-A、B、DRB1等位基因型及单倍型分布具有典型南方人群的特点,此资料有助于为临床移植寻找合适匹配的供受对.