A novel splice site variant in the POPDC3 causes autosomal recessive limb-girdle muscular dystrophy type 26.

Limb-Girdle muscular dystrophy (LGMD) is a group of muscle disorders with highly heterogeneous genetic patterns and clinical phenotypes, and this group includes multiple subtypes. Different LGMD subtypes have similar phenotypes and clinical overlaps, these subtypes are difficult to distinguish by clinical symptoms alone and can only be accurately diagnosed by analysis in combination with definitive genetic test results. Here, we report a female presenting features of LGMD. After analysis of whole-exome sequencing data, a novel homozygous POPDC3 variant c.486-1G>A (rs113419658) located in the acceptor splice site of intron 2 was identified in the proband. The variant effect on splicing were analyzed by genetic analysis based on cDNA synthesized by the patient's RNA. cDNA analysis indicated that the novel homozygous POPDC3 splice variant disrupted original acceptor splice site, which can cause a frameshift in the mRNA of the POPDC3 gene, thereby producing a truncated POPDC3 protein and ultimately affecting its normal function. POPDC3 variant was recently associated with recessive limb-girdle muscular dystrophy type 26 (LGMDR26). Based on the above results, we hypothesize that this variant is probably a pathogenic variant, and expand the gene variant spectrum of POPDC3.

Association between gene polymorphisms of voltage-dependent Ca2+ channels and hypertension in the Dai people of China: a case-control study.

BACKGROUND: Abnormal calcium homeostasis related to the development of hypertension. As the key regulator of intracellular calcium concentration, voltage-dependent calcium channels (VDCCs), the variations in these genes may have important effects on the development of hypertension. Here we evaluate VDCCs variability with respect to hypertension in the Dai ethnic group of China. METHODS: A total of 1034 samples from Dai individuals were collected, of which 495 were used as cases, and 539 were used as controls. Blood pressure was measured using a standard mercury measurement method, three times with a rest for 5 min, and the average was used for analyses. Seventeen single nucleotide polymorphisms (SNPs) in the four protein-coding genes (CACNA1A, CACNA1C, CACNA1S, CACNB2) of VDCCs were identified by multiplex PCR-SNP typing technique. Chi-square tests and regression models were used to analyse the associations of SNPs with hypertension. RESULTS: The results of chi-square tests showed that the allele frequencies of 5 SNPs were significantly different between the case and the control groups (P < 0.05), but the statistical significance was lost after Bonferroni's correction. However, after adjusting for BMI, age, sex and other factors by logistic regression analyses, the results showed that 5 SNPs consistent with chi-square tests (rs2365293, rs17539088, rs16917217, rs61839222 and rs10425859) were still statistically positive. CONCLUSIONS: This finding suggested that the significant association of these SNPs with hypertension may be noteworthy in future studies.

Characterization of a novel echovirus 21 strain isolated from a healthy child in China in 2013.

Echovirus 21 (E21) belongs to the species Enterovirus B, whose members are frequently associated with acute flaccid paralysis. E21 strain 553/YN/CHN/2013 was isolated from a healthy child in Yunnan, China, in 2013. This is the first report of the complete genome sequence of E21 in China. This strain shared 81.7% nucleotide sequence identity and 96.8% amino acid sequence identity with the E21 prototype strain Farina. Although strain 553/YN/CHN/2013 belongs to the E21 serotype, the only similarity to the E21 strain was in the VP1 region, as other genomic regions, including VP2-VP4, were more similar to other EV-B members. Recombination analysis showed evidence of recombination events between E21 and other EV-B viruses. E21 strain 553/YN/CHN/2013 failed to infect suckling mice via intracerebral injection. Surveillance of E21 is very important to help forecast the potential of emerging E21 outbreaks and related diseases.

Molecular characterization of Coxsackievirus A16 strains isolated from children with severe hand, foot, and mouth disease in Yunnan, Southwest China, during 2009-2015.

Coxsackievirus A16 (CV-A16) commonly causes mild symptoms, but severe diseases, such as aseptic meningitis, encephalitis, and even fatal cases, have been reported. Thirteen CV-A16 strains were isolated from patients with severe hand, foot, and mouth disease in Yunnan, Southwest China, from 2009 to 2015. Subgenotype B1a and B1b of CV-A16 were predominantly circulating the region with B1b the predominant strain in recent years. The mean rate of nucleotide substitution based on the VP1 gene sequence was 4.545 × 10 -3 substitution per site per year from 2009 to 2015. These results may help in understanding the genetic diversity of CV-A16 and develop a CV-A16 vaccine.

Molecular evolution of two asymptomatic echovirus 6 strains that constitute a novel branch of recently epidemic echovirus 6 in China.

BACKGROUND: Echovirus 6 (E6) infections are associated with aseptic meningitis and acute flaccid paralysis (AFP). But some infections, sometimes most of them, are asymptomatic. The mechanism of E6 virulence is unknown. Analyses of the molecular evolution of asymptomatic E6 may help understand why the infections show different manifestations. METHODS: Ninety-six stool samples of healthy children in Yunnan, China were collected and two E6 strains were isolated from them. The whole genomes of these two E6 strains were sequenced, and their molecular evolution was analyzed. RESULTS: The results showed that the two E6 strains may be derived from KJ7724XX strains, which were predominant in AFP patients in Shangdong in 2011. The evolution was accelerated when the two E6 strains formed, although no positive selection site was found. The 11 exclusive mutations on which selection force significantly changed were found in the 2C, 3AB and 3C genes. CONCLUSION: There are some E6 strains which did not cause the disease in the children of Yunnan. These E6 strains maybe come from a recombinant E6 strain which was associated with the outbreak of AFP in Shangdong in 2011. However, some new mutations were found in the 2C, 3AB and 3C genes of these asymptomatic strains, and these mutations may be constraint by the natural selection and could be potentially responsible for clinical presentations.

Mitochondrial haplogroup M9a1a1c1b is associated with hypoxic adaptation in the Tibetans.

While hypoxic environment at high altitude remains a major challenge for travelers from low-altitude areas, Tibetans have adapted to the high-altitude environment. Mitochondria are the energy conversion and supplement centers in eukaryotic cells. In recent years, studies have found that the diversity of the mitochondrial genome may have a role in the adaptation to hypoxia in Tibetans. In this study, mitochondrial haplogroup classification and variant genotyping were performed in Tibetan and Han Chinese populations living at different altitudes. The frequencies of mitochondrial haplogroups B and M7 in the high-altitude population were significantly lower compared with those in the low-altitude population (P=0.003 and 0.029, respectively), whereas the frequencies of haplogroups G and M9a1a1c1b in the high-altitude group were significantly higher compared with those in the low-altitude group (P=0.01 and 0.002, respectively). The frequencies of T3394C and G7697A, which are the definition sites of haplogroup M9a1a1c1b, were significantly higher in the high-altitude group compared with that in the low-altitude group (P=0.012 and 0.02, respectively). Our results suggest that mitochondrial haplogroups B and M7 are associated with inadaptability to hypoxic environments, whereas haplogroups G and M9a1a1c1b may be associated with hypoxic adaptation. In particular, the T3394C and G7697A variants on haplogroup M9a1a1c1b may be the primary cause of adaptation to hypoxia.

Gene frequency and haplotype distribution of hemoglobin E among seven minority groups of Yunnan, China.

OBJECTIVE: The aim of this study was to determine the distribution and origin of hemoglobin E (HbE) in seven minority groups from various geographical regions of the malaria-endemic Yunnan province, southwestern China, which have similar ethnic origins and geographic relationships with HbE-prevalent populations of Southeast Asian countries. METHODS: By using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) methods, the prevalence of HbE was examined in 1488 individuals from seven native minority groups of Yunnan, and ß-globin gene cluster haplotypes were determined on 1420 chromosomes. RESULTS: The prevalence of HbE in the study populations ranged from 1.5 to 39.1%. Higher HbE prevalence was correlated with the minority groups of Tibeto-Burman origin and groups from the Dehong district. The ßE -globin genes in Yunnan were mostly associated with three haplotypes [-+++++-], [+----+-], and [-+-+++-] on chromosomes with gene framework 2. Interestingly, the predominant ßE associated haplotype in Yunnan minorities was remarkably different from that in other previously reported populations. This study, for the first time, reports population-based data on the heterogeneity of HbE gene frequencies and haplotype distribution in native minorities from southwestern China. CONCLUSIONS: Natural selection based on the presence of malaria, ethnic origin, and epistatic interactions may be factors of varying importance for the remarkable variation in HbE frequency among these minority groups. In addition, there appears to be a common origin of the ßE -globin gene in populations from Yunnan and Southeast Asia. Am. J. Hum. Biol. 28:927-931, 2016. © 2016Wiley Periodicals, Inc.

[Impact of natural selection on the polymorphism of HLA-G 3'UTR among five ethnic Chinese populations].

OBJECTIVE: To assess the impact of natural selection and genetic background on the polymorphisms of HLA-G 3-untranslated regions (UTR) among five ethnic Chinese populations. METHODS: PCR and DNA sequencing were used to determine the polymorphisms among 432 individuals from the five ethnic populations. Their genetic background was determined by genotyping of 10 short tandem repeats (STRs). RESULTS: Eight variations were identified among Gelao, Mongolian and Kirgiz populations, while only 7 were found in Shui and Dai people. For all 3 southern populations (Gelao, Shui, and Dai), the observed heterozygosites (Ho) was higher than expected heterozygosities (He). But this was reversed for the 2 northern populations (Mongolian and Kirgiz). The Ho and He of the 10 neutral STRs were in random distribution. Ewens-Watterson testing based on haplotypes of the HLA-G 3'UTR has suggested that a natural selection had occurred in the region where Dai and Shui had inhabited, but not in the northern region where Mongolian and Kirgiz population inhabited. Polygenetic trees based on the HLA and STRs were also different. CONCLUSION: The HLA-G 3'UTR of Dai and Shui people who lived in southern China may have subjected to a selection pressure. Based on current knowledge, this pressure may have been driven by a pathogenic selection.

[Association study of telomere length with idiopathic male infertility].

Telomeres are evolutionary conserved, multifunctional DNA-protein complexes located at the ends of eukaryotic chromosomes. Telomeres maintain chromosome stability and genome integrity and also play an important role in meiosis which aid in synapsis, homologous recombination, and segregation. Sperm telomere has been reported to play an important role in fertilization and embryo development. Nowadays, the association between telomere and reproduction is one of the major areas of interest, however whether sperm telomere associated with male infertility is not clear. In this study, in order to find out the association between Chinese idiopathic infertility and sperm telomere length, we analyzed the difference of sperm telomere length between idiopathic infertile men and normal fertile men, as well as the correlations between sperm telomere length and human semen characteristics. We analyzed 126 Chinese idiopathic infertile men and 138 normal fertile men for sperm telomere length by using quantitative PCR. We found that the relative sperm mean telomere length of infertile men was significantly shorter than that of fertile men (2.894 ± 0.115 vs. 4.016 ± 0.603, P=5.097 x 10⁻5). Both sperm count and semen progressive motility are related with telomere length. Our results suggest that sperm telomere length is associated with idiopathic male infertility of China and we proposed the possibility that shorter telomeres in sperm chromosome will reduce spermatogenesis and sperm functions, which finally affected the fertility of male.

Determination of the 3'UTR region of HLA-B*53:01:03 sequence by next generation sequencing.

The 3'UTR of the HLA-B*53:01:03 allele has been determined by next generation sequencing.

Association study of FLT4 and HYDIN single nucleotide polymorphisms with atrial septal defect susceptibility in the Han Chinese population of Southwest China.

BACKGROUND: Atrial septal defect (ASD) is a common form of congenital heart disease. Although several genes related to ASD have been found, the genetic factors of ASD remain unclear. This study aimed to evaluate the correlation between 10 candidate single nucleotide polymorphisms (SNPs) and sporadic atrial septal defects. METHODS: Based on the results of 34 individual whole exome sequences, 10 candidate SNPs were selected. In total, 489 ASD samples and 420 normal samples were collected. The 10 SNPs in the case group and the control group were identified through Snapshot genotyping technology. The χ2-test and unconditional regression model were used to evaluate the relationship between ASD and each candidate SNP. Haploview software was used to perform linkage disequilibrium and haplotype analysis. RESULTS: The χ2 results showed that the FLT4 rs383985 (P = 0.003, OR = 1.115-1.773), HYDIN rs7198975 (P = 0.04621, OR = 1.003-1.461), and HYDIN rs1774266 (P = 0.04621, OR = 1.003-1.461) alleles were significantly different between the control group and the case group (P < 0.05). Only the association with the FLT4 polymorphism was statistically significant after adjustment for multiple comparisons. CONCLUSION: These findings suggest that a possible molecular pathogenesis associated with sporadic ASD is worth exploring in future studies.

Correlation between the linguistic affinity and genetic diversity of Chinese ethnic groups.

As the world's most populous nation, China exhibits a population with 56 nationalities. We already know the associations between genetic relationship of these ethnic groups in China and their geographic distributions are closely. However, the correlations between genetic diversity and linguistic affinities have still not been fully revealed in China. To investigate these correlations, 31 populations and 1527 samples were chosen, and the languages of this population covered all of the languages spoken in mainland China (including 8 main linguistic families and 16 subfamilies). The genetic polymorphisms of the populations were investigated using 10 autosomal microsatellites. Five ethnic groups, which included 234 samples, were genotyped in this survey, and the data collected from the other 26 populations were obtained from our previous study. An analysis of molecular variance, principal coordinate analysis, clustering analysis using the STRUCTURE and the Mantel test were used to investigate the correlations between genetic diversity and linguistic affinity. These analyses indicated that most populations who speak the same language demonstrate a similar genetic composition, although a few populations deviated from this linkage between genetics and language. The demographic histories of these populations who deviated from this linkage were investigated. Obvious reasons for why evolutionary processes of genetics and linguistics separated in these populations included geographic isolation, gene replacement, language replacement and intermarriage. Thus, we proposed that the consistency of genetic and linguistic evolution is still present in most populations in China; however, this consistency can be broken by many factors, such as isolation, language replacement or intermarriage.

[Association of polymorphisms of PTPN22 and PADI4 genes with rheumatoid arthritis in Yunnan].

OBJECTIVE: To assess the association between genetic polymorphisms of 7 SNPs in PTPN22 and PADI4 genes and susceptibility to rheumatoid arthritis in Yunnan. METHODS: A case-control study was carried out on 192 patients of rheumatoid arthritis and 288 healthy controls. Genotypes of rs33996649 and 1858 loci within PTPN22 gene, and rs11203366 and rs874881 loci within PADI4 gene were determined with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Genotypes of rs1635579, rs2428736 and rs2240340 in PADI4 gene were determined with pyrosequencing. RESULTS: The frequencies of alleles and genotypes of rs2240340 locus in PADI4 gene showed a significant difference between rheumatoid arthritis and controls in Yunnan population (P U+003C 0.05). CONCLUSION: Our results suggested that rs2240340 in PADI4 gene is associated with susceptibility to rheumatoid arthritis in Yunnan.

[Distribution of HLA-G 14 bp insertion/deletion polymorphism and their associations with HLA-A alleles in four Chinese ethnic groups].

Recently, a 14 bp insertion/deletion polymorphism (+14 bp/-14 bp) in exon 8 of the human leucocyte antigen-G (HLA-G) gene has been widely recognized to associate with recurrent miscarriage, autoimmune diseases, hepatocellular carcinoma and other diseases. Our previous studies have shown the distribution characteristics of linguistic family for HLA-G 14 bp insertion/deletion in different ethnic groups. In the present study, we investigated the distribution of HLA-G 14 bp insertion/deletion polymorphism and their subsequent associations with HLA-A alleles in Tu, Yugu, Lisu and Nu ethnic populations based upon the HLA-A genotyping data. Our results showed that the frequencies of the 14 bp insertion/deletion polymorphism were diverse in these four populations while that in the same linguistic subfamily was similar. The significant difference in different linguistic subfamily except for Han and Mongolian language subfamily was identified. In addition, the 14 bp insertion was found to associate with HLA-A alleles in different ethnic populations.

[Association between six single nucleotide polymorphisms of EGLN1 gene and adaptation to high-altitude hypoxia].

To investigate the association between SNPs located in 5'UTR and intron of prolyl hydroxylase 2 (EGLN1 or PHD2) and adaptation to high-altitude hypoxia, the SNPs (rs2066140, rs2808584, rs2491405, rs2486741, rs2486734 and rs21533646) of EGLN1 gene were genotyped using Sequenom MassArray genotyping system in 152 unrelated healthy Tibetan individuals (3 650 m altitude) and 192 Han (5 00 m altitude), and the haplotypes of these SNPs were constructed and analyzed. Our results showed all the homozygous genotypes of six SNPs loci were significantly different between the two groups (P<0.05). The frequencies of haplotypes G-G (rs2066140 and rs2808584) and G-C (rs2486741 and rs2486734) of high-altitude group were significantly different from low-altitude group (P<0.05). In addition, the frequencies of haplotypes C-A (rs2066140 and rs2808584) and C-T (rs2486741 and rs2486734) of high-altitude group were significantly lower than those in low-altitude group (P<0.05). Our results indicate that the polymorphism of homozygous genotype in six SNPs and their haplotypes were associated with adaptation to high-altitude hypoxia.

Comprehensive research on the distribution of HLA-DRB1 in Chinese populations.

By presenting antigen peptides, HLA-DRB1 plays an important role in the immune system. However, the allele frequency of HLA-DRB1 exon 2 across China has not been comprehensively studied, especially in minority populations. We sampled 3757 individuals from 59 population. The HLA-DRB1 region from 212 to 463 bp (NM_002124.4 exon 2) in each population was sequenced by Sanger sequencing and genotyped via SBTengine® software, and the allele frequency was calculated by GenAlEx 6.5. Eighty-two DRB1 alleles were identified. The expected heterozygosity of DRB1 was lower in the south than in the north, which was inconsistent with the Y chromosome and mitochondrial DNA results. The Mantel test and nonparametric correlation analysis showed that the correlations of the genetic distance with geographical distance and of DRB1 allele frequencies with latitude weakened after the southern and northern groups were considered separately. Principal coordinate analysis showed that populations speaking the same languages were not codistributed. Compared with other genetic markers, the distribution of DRB1 seems less affected by geographic distance and ethnic origin. Local factors such as gene flow with neighbouring populations, geographic isolation or natural selection are important forces shaping the DRB1 gene pool of local populations.

[Association between gene polymorphisms and myocardial infarction in Han Chinese of Yunnan province].

OBJECTIVE: To determine frequencies of genetic polymorphisms of coagulation factor VII (FVII), coagulation factor FXII (FXII), fibrinogen (FBG) and 9p21 in ethnic Han Chinese from Yunnan province, and to assess the association between such polymorphisms and onset of myocardial infarction (MI). METHODS: One hundred and forty-two patients with MI and 192 healthy controls were analyzed. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and pyrosequencing were used to determine the genotypes of FVII, FXII, FBG and 9p21. RESULTS: No significant difference was found in the frequencies of R353Q, 5'F7, C46T, -148C/T, rs1333049 and rs4977574 loci between the two groups (P> 0.05). However, the frequencies of AA of -455G/A, T and TT of rs1333040, T and TT of rs10116277 and G and GG of rs2383207 were significantly higher in MI group compared with the controls (P< 0.05), whilst the frequencies of CT of rs1333040 and GT of rs10116277 were significantly lower in MI group compared with the controls (P<0.05). CONCLUSION: Polymorphisms of FVII, FXII, -148C/T of FBG and rs1333049 of 9p21 were not associated with myocardial infarction. Polymorphisms of -455G/A of FBG and rs1333040, rs10116277 and rs2383207 of 9p21 may be associated with MI in ethnic Han Chinese from Yunnan province.

[Detection and preliminary study of a family carrying a CCR5Δ32 deletional mutation].

OBJECTIVE: To investigate the frequencies of chemokine (C-C motif) receptor 5 gene (CCR5)Δ32 deletional mutation of in Han and Dai populations from Yunnan province. Immortalized cell lines were derived from a family carrying the CCR5Δ32 mutation. METHODS: Blood samples of 346 Han and 355 Dai individuals were collected for genotyping. The coding regions of CCR5 gene were amplified with PCR followed by agarose gel electrophoresis. Suspected mutations were verified with DNA sequencing. Immortalized cell lines were constructed by using Epstain Barr virus and cyclosporine A. The difference between the cell lines and original blood samples was verified with PCR. RESULTS: One ethnic Han individual was confirmed to be heterozygous for a deletional mutation by sequencing, which has led to discovery of a family with CCR5Δ32. Nine immortalized cell lines were established from this family, and no difference between the cell lines and original blood samples was detected by PCR. CONCLUSION: Together with previous reports, this study has indicated a significant difference in CCR5Δ32 among different ethnic groups in China. Established immortalized cell lines can also provide material for future research.

[Association between Alu insertion polymorphisms and HLA class I alleles in Chinese Lisu and Nu ethnic populations].

OBJECTIVE: To investigate the frequencies of HLA-Alu repeat polymorphisms (AluMICB, AluTF, AluHJ, AluHG and AluHF) in Chinese Lisu and Nu ethnic populations. METHODS: The frequencies of HLA-Alu repeat polymorphisms in above populations were determined with polymerase chain reaction (PCR). The associations between HLA-Alu repeat polymorphisms and HLA-A, HLA-B and HLA-C alleles were also analyzed. Phylogenetic trees were constructed with genetic distance calculated from the frequencies of HLA-Alu repeat polymorphisms. RESULTS: Frequencies of AluTF*2 and AluHF*2 were different between the two populations (P< 0.05), while those of other three insertions were similar. The strength of association between HLA-Alus and HLA alleles were different (P< 0.05) in the two populations. Although AluMICB*2 were associated with HLA-B*56:01 in both populations, the association was stronger in Lisu population (74.0%) but moderate in Nu population (30.7%). HLA-Alus were associated with particular HLA subtypes, e.g., AluHG*2 with certain HLA-A*02 subtypes. By phylogenetic analysis, Lisu and Nu were clustered together with southern Chinese and Thai populations. CONCLUSION: The distribution of HLA-Alus and the strength of associations between HLA-Alus and HLA class I alleles have varied between the two populations. Study of this association may facilitate identification of origins, evolution, progenitor haplotypes and recombination within the HLA class I region.

Diversity of killer cell immunoglobulin-like receptor genes in four ethnic groups in China.

Killer cell immunoglobulin-like receptors (KIRs) show extensive variation in terms of gene content and allelic polymorphisms among different populations. The aim of this study was to analyze the distribution of KIR genes in the Bulang, Nu, Yugu, and Zhuang ethnic groups, which belong to four different language families in China, and thus to provide basic KIR gene and genotype data for these Chinese ethnic groups. Genotyping of 16 KIR genes was performed in 425 unrelated individuals using the polymerase chain reaction-sequence-specific oligonucleotide probe method with the Luminex MultiAnalyte Profiling System. The four framework KIR genes were detected in all four ethnic groups. The activating KIR genes as well as the inhibitory KIR genes showed extreme diversity among these four populations. A total of 35 distinct KIR genotypes were identified, one of which was previously unknown. The four most common genotypes were identified in all four populations and comprised 66.1~91.1% of all the genotypes. The group A haplotype occurred more frequently than the group B haplotype in the Nu, Yugu, and Zhuang populations, as in other East Asian populations. In contrast, the group A and group B haplotypes occurred equally in the Bulang population. The results of the present study suggested that the KIR genes and genotypes are diverse in these four ethnic groups, and each ethnic group has its own characteristic KIR distribution. The findings with respect to KIR gene diversity in these four populations should provide relevant genomic diversity data for the future study of viral infections, autoimmune diseases, and reproductive fitness.