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Background: To evaluate the prevalence of alpha- and beta-thalassemia in Ji'an City, Jiangxi Province, 28,941 people in the region were genetically screened to identify various thalassemia genotypes. Methods: High-throughput amplicon sequencing and gap-PCR was used to screen 301 thalassemia alleles in 28,941 people in the region. Pregnant women were the focus of this screening, and if a pregnant woman harbored mutations in a thalassemia-inducing gene, her spouse was also genetically tested. Results: Of the participants, 2,380 people were carriers of thalassemia, with at least one thalassemia allele, including 1,742 alpha-thalassemia carriers, 686 beta-thalassemia carriers and 48 composite alpha- and beta-thalassemia carriers. The total carrying rate of thalassemia in Ji'an was 8.22%, and the carrying rates of alpha- and beta-thalassemia were 6% and 2.37%, respectively. In addition, the first measured carrier rate of composite alpha- and beta-thalassemia in Ji'an was 0.17%. According to the geographical distribution of the 1,742 alpha -thalassemia carriers, the city with the highest carrier rate was Suichuan, followed by Wan’an and Taihe. According to the geographical distribution of the 686 beta-thalassemia carriers, the top three cities with high carrier rates were Suichuan, Wan'an and Xiajiang, sequentially. Conclusions: This research emphasizes the importance of large-scale population screening and that comprehensive molecular epidemiology data are necessary for the proper prevention and treatment of thalassemia. The epidemiological data updated in this research may enable the local government to focus on the severity of this disease and determine a method for effective resource allocation under limited resource conditions.

To explore the cause of abortion and strategy of prenatal diagnosis for pregnant women with high risk for chromosomal abnormalities by using copy number variation sequencing (CNV-seq) and short tandem repeats (STR) analysis.A total of 36 samples were collected, including amniotic fluid, abortion tissue, whole blood, chorionic villi and umbilical cord blood. CNV-seq and STR analysis were carried out to detect microdeletions, microduplications, chromosomal aneuploidies, mosaicisms and triploidies.Among all samples, 1 was detected with 4p15.1p16.3 and 14q11.1q22.1 duplication, 1 was detected with 19p13.3 deletion, 8 were detected with chromosomal aneuploidies, 4 were detected with mosaicisms, two were detected with triploidies. No definite pathogenic CNVs were detected in 20 samples, which yielded a positive detection rate of 44.44%.As a high-throughput detection method, CNV-seq has the advantages of rapidity, simplicity and high accuracy. It may suit prenatal diagnosis and analysis of abortion factors in combination with STR analysis.

Background Oculocutaneous albinism (OCA) is a group of rare genetically heterogeneous disorders. The present study aimed to identify the genetic cause of a Chinese Han family with non-syndromic oculocutaneous albinism (OCA). Case presentation Here, we report an 11-month-old male proband from a Chinese Han non-consanguineous family, who presented with milky skin, yellow white hair, nystagmus, astigmatism, and hypermetropia. We performed the targeted next-generation sequencing (NGS) on the proband and identified two novel compound heterozygous variants (c.1865 T > C (p.Leu622Pro) and exons 17–21 deletion) in OCA2 gene associated with OCA type 2 (OCA2, OMIM 203200). Meanwhile, a previously reported heterozygous mutation (c.4805G > A) in MYO7 gene related with Usher syndrome type 1B was found. The online tools SIFT, PolyPhen-2, and Mutation Taster predicted variant c.1865 T > C was probably damaging. The residue p.Leu622 was in a highly conserved region among species by CLUSTALW. Three-dimensional homology model with I-TASSER indicated that p.Leu622Pro variant disturbed the formation of the α-helix, resulting in a random coil structure. The gross deletion (exons 17–21) in OCA2 gene has was not been reported previously. These two novel variants in OCA2 gene were inherited from each parent respectively, after verification by Sanger sequencing and quantitative PCR (qPCR) in the family. Conclusions This study indicates the two novel compound heterozygous mutations in OCA2 gene may be responsible for clinical manifestations of OCA2. It expands the mutation spectrum of OCA2 gene and is helpful to screen for large deletions with targeted NGS protocol in monogenic disease. It also assists the genetic counselling, carrier screening and personalized healthcare of the disease. Electronic supplementary material The online version of this article (10.1186/s12881-019-0850-7) contains supplementary material, which is available to authorized users.

Most of the variation in the human genome is a single nucleotide variation (SNV) based on a single base or small fragment insertions and deletions and genomic copy number variation (CNV). Both types of mutations are involved in many human diseases. Such diseases often have complex clinical symptoms and difficult clinical diagnosis, so an effective detection method is needed to help clinical diagnosis and prevent birth defects. With the development of sequencing technology, the method of chip capture combined with high-throughput sequencing has been extensively used because of its high throughput, high accuracy, high speed and low cost. This study designed a chip that captures the coding region of 3043 genes associated with 4013 monogenic diseases. In addition, 148 chromosomal abnormalities can be identified by setting targets in specific regions. Compared with the whole exon chip, the chip can detect 4013 monogenic diseases and 148 chromosomal abnormalities at a lower cost, including SNV, intra-gene CNV and genomic copy number variation. This study utilized a strategy of combining the BGISEQ500 sequencing platform with the chip to identify 102 disease-associated mutations in 63 patients, 69 of which were new mutations. The evaluation test results also show that this combination complies with the requirements of clinical testing and has good clinical application value.

Supplemental Digital Content is available in the text, Hearing loss is a highly heterogeneous disease presented with various phenotypes. Genetic testing of disease-causing mutations plays an important role in precise diagnosis and fertility guidance of heredity hearing loss. Here we reported an effective method employing target enrichment and BGISEQ-500 platform to detect clinically relevant alterations for heredity hearing patients in a single assay. In this study, we designed an array based chip, containing 127 genes related to hearing loss. Then we conducted targeted next-generation sequencing toward 58 patients to make a precise diagnosis using BGISEQ-500 platform. We successfully detected disease-causing mutations in 77.59% (45/58) of the patients with hearing loss. Finally, a total of 62 disease-causing mutations were identified, including 31 missense, 17 Indel, 11 splicing, 2 synonymous, and 1 copy number variant. 58.06% (36/62) of which has never been reported before. To our knowledge, this is the first report using BGISEQ-500 platform to investigate both syndromic and nonsyndromic hearing loss in the Chinese population. The results showed that this method can greatly assist and enhance hearing loss diagnosis and improve molecular diagnostics outcome.

The list of 54 genes causing hereditary eye diseases. (DOCX 19Â kb)

PurposeCardiovascular diseases are the most common cause of death globally. In which atrioventricular block (AVB) is a common disorder with genetic causes, but the responsible genes have not been fully identified yet. To determine the underlying causative genes involved in cardiac AVB, here we report a three-generation Chinese family with severe autosomal dominant cardiac AVB that has been ruled out as being caused by known genes mutations.MethodsWhole-exome sequencing was performed in five affected family members across three generations, and co-segregation analysis was validated on other members of this family.ResultsWhole-exome sequencing and subsequent co-segregation validation identified a novel germline heterozygous point missense mutation, c.49287C>A (p.N16429K), in the titin (TTN, NM_001267550.2) gene in all 5 affected family members, but not in the unaffected family members. The point mutation is predicted to be functionally deleterious by in-silico software tools. Our finding was further supported by the conservative analysis across species.ConclusionBased on this study, TTN was identified as a potential novel candidate gene for autosomal dominant AVB; this study expands the mutational spectrum of TTN gene and is the first to implicate TTN mutations as AVB disease causing in a Chinese pedigree.

Background Cardiovascular diseases are the most common cause of death globally. In which atrioventricular block (AVB) is a common disorder with genetic causes, but the responsible genes have not been fully identified yet. To determine the underlying causative genes involved in cardiac AVB, here we report a three-generation Chinese family with severe autosomal dominant cardiac AVB that has been ruled out as being caused by known genes mutations. Methods Whole-exome sequencing was performed in five affected family members across three generations, and co-segregation analysis was validated on other members of this family. Results Whole-exome sequencing and subsequent co-segregation validation identified a novel germline heterozygous point missense mutation, c.49287C > A (p.N16429K), in the titin (TTN, NM_001267550.2) gene in all 5 affected family members but not in the unaffected family members, neither in the large population according to the Genome Aggregation Database ( https://gnomad.broadinstitute.org/ ). The point mutation is predicted to be functionally deleterious by in-silico software tools. Our finding was further supported by the conservative analysis across species. Conclusion Based on this study, TTN was identified as a potential novel candidate gene for autosomal dominant AVB; this study expands the mutational spectrum of TTN gene and is the first to implicate TTN mutations as AVB disease causing in a Chinese pedigree.

Syndactyly is one of the most frequent hereditary limb malformations with clinical and genetical complexity. Autosomal dominant Syndactyly type IV (SD4) is a very rare form of syndactyly, occurring as a result of heterozygous mutation in an SHH regulatory element (ZRS) that resides in intron 5 of the LMBR1 gene on chromosome 7q36.3. The SD4 is characterized by complete cutaneous syndactyly of all fingers, cup-shaped hands due to flexion of the fingers and accompanied by polydactyly. Here, we firstly reported a big Chinese family, manifesting cup-shaped hands consistent with SD4 and intrafamilial heterogeneity in clinical phenotype of tibial and fibulal shortening, triphalangeal thumb-polysyndactyly syndrome (TPTPS). Genetically, we identified a novel duplication of ∼222.23 kb covering exons 2-17 of the LMBR1 gene in this family by next generation sequencing. This case expands our new clinical understanding of SD4 phenotype.

With the development of next generation sequencing, more and more common inherited diseases have been reported. However, accurate and convenient molecular diagnosis cannot be achieved easily because of the enormous size of disease causing mutations. In this study, we introduced a new single-step method for the genetic analysis of patients and carriers in real clinical settings. All kinds of disease causing mutations can be detected at the same time in patients with Mendelian diseases or carriers. First, we evaluated this technology using YH cell line DNA and 9 samples with known mutations. Accuracy and stability of 99.80% and 99.58% were achieved respectively. Then, a total of 303 patients were tested using our targeted NGS approaches, 50.17% of which were found to have deleterious mutations and molecular confirmation of the clinical diagnosis. We identified 219 disease causing mutations, 43.84% (96/219) of which has never been reported before. Additionally, we developed a new deleteriousness prediction method for nonsynonymous SNVs, and an automating annotation and diagnosis system for Mendelian diseases, thus greatly assisting and enhancing Mendelian diseases diagnosis and helping to make a precise diagnosis for patients with Mendelian diseases.

The genetic basis of congenital mental retardation includes chromosomal anomalies and single gene mutations. In addition to chromosome microarray analysis, next‑generation sequencing (NGS) and Sanger sequencing have additionally been applied to identify single gene mutations. However, no methods exist to identify the cause of an anomaly in one step. The present study applied an improved targeted NGS method to diagnose an 8‑year‑old Chinese Han female with mental retardation in one step. The microdeletion 17p11.2 was successfully detected by the improved targeted NGS and no single gene mutations were identified. The same microdeletion was verified using low coverage whole‑genome sequencing. Fertility guidance was also given to the patient's parents. In the present study, an improved targeted NGS method was applied to diagnose non‑syndromic mental retardation of unknown cause in one step. This improved method has the potential to be developed into a screening panel for the effective diagnosis of genetic abnormalities in non‑syndromic mental retardation and other congenital anomalies.

Mutations in the LAMA2 gene cause laminin α‑2 (merosin)‑deficient congenital muscular dystrophies, which are autosomal recessive muscle disorders. Laminin α‑2 is widely expressed in the basement membrane of skeletal muscle, the myotendinous junctions and extra‑synaptically at neuromuscular synapses. In the present study, target next‑generation sequencing was used for mutation detection, and polymerase chain reaction (PCR) analysis and Sanger sequencing were used in the identification of small deletions. Subsequently, quantitative PCR (qPCR) was performed to characterize the identified deletion encompassing exon five of the LAMA2 gene. Two causative mutations were identified using target region sequencing which provided the additional information required to facilitate clinical diagnosis. One heterozygous mutation (p. Lys682LysfsX22) was identified and confirmed by Sanger sequencing, and another heterozygous mutation (Exon5del) was found and validated by qPCR. Co‑segregation analysis indicated that the Exon5del mutation originated from the proband's mother and the previously reported frameshift mutation (p. Lys682LysfsX22) was inherited from the proband's father. To the best of our knowledge, the present study was the first to report an entire exon five deletion in the LAMA2 gene.

A new xylanase gene, named xyn186, was cloned by the genome-walking PCR method from the Alternaria sp. HB186. The sequence of xyn186 contains a 748 bp open reading frame separated by one intron with the size of 52 bp. The cDNA was obtained by DpnI-mediated intron deletion. The cDNA was cloned into pHBM905A and transformed into Pichia pastoris GS115 to screen xylanase-secreting transformants on RBB-xylan plates. The molecular mass of the enzyme was estimated to be 23 kDa on SDS-PAGE. The optimal pH and temperature of the purified enzyme is 6 and 50°C, respectively. The K (m) and V (max) valued for birchwood xylan are 1.404 mg ml(-1) and 0.2748 mmol min(-1) mg(-1), respectively. The inhibitory effects of various metal ions were investigated, Cu(2+) and Hg(2+) ions inhibited most of the enzyme activity. The gene copy number of xyn186 in the genome of P. pastoris was estimated as two by the Real-time PCR. To date, xyn186 gene is the first xylanase gene cloned from the genus Alternaria.

Background: The conventional degumming process of ramie with alkaline treatment at high temperature causes severe environmental pollution. Pectate lyases can be used to remove pectin from ramie in a degumming process with reduced environmental pollution and energy consumption. Pectate lyase PEL168 from Bacillus subtilis has been previously characterized and the protein structure was resolved. However, Bacillus is not a suitable host for pectate lyases during the degumming process since most Bacillus produce cellulases endogenously with a detrimental effect to the fiber. Pichia pastoris, which does not express endogenous cellulases and has high secretion capability, will be an ideal host for the expression. No previous work was reported concerning the heterologous expression of pectate lyase PEL168 in P. pastoris with an aim for industrial application in ramie bio-degumming. Results: The gene pel168 was expressed in P. pastoris in this study. The recombinant protein PEL168 in P. pastoris (PEL168P) showed two bands of 48.6 kDa and 51.4 kDa on SDS-PAGE whereas the enzyme expressed in E. coli (PEL168E) was the same as predicted with a band of 46 kDa. Deglycosylation digestion suggested that PEL168P was glycosylated. The optimum reaction temperature of the two PEL168s was 50°C, and the optimum pH 9.5. After preincubation at 60°C for 20 min, PEL168E completely lost its activity, whereas PEL168P kept 26% of the residual activity. PEL168P had a specific activity of 1320 U/mg with a Km of 0.09 mg/ml and a Vmax of 18.13 μmol/min. K + , Li + ,N i 2+ and Sr 2+ showed little or no inhibitory effect on PEL168P activity, and Ca 2+ enhanced enzyme activity by 38%. PEL168P can remove the pectin from ramie effectively in a degumming process. A 1.5 fold increase of PEL168 enzyme expression in P. pastoris was achieved by further codon optimization. Conclusions: Pectate lyase PEL168 with an available protein structure can be heterologously expressed in P. pastoris. The characterized recombinant PEL168P can be used to remove pectin from ramie efficiently and the expression level of PEL168 in P. pastoris was increased markedly by codon optimization. Therefore, PEL168 is an ideal candidate for further optimization and engineering for bio-degumming.

Alkaliphilic and halophilic Bacillus sp. BG-CS10 was isolated from Zabuye Salt Lake, Tibet. The gene celB, encoding a halophilic cellulase was identified from the genomic library of BG-CS10. CelB belongs to the cellulase superfamily and DUF291 superfamily, with an unknown function domain and less than 58% identity to other cellulases in GenBank. The purified recombinant protein (molecular weight: 62 kDa) can hydrolyze soluble cellulose substrates containing beta-1,4-linkages, such as carboxylmethyl cellulose and konjac glucomannan, but has no exoglucanase and β-glucosidase activities. Thus, CelB is a cellulase with an endo mode of action and glucomannanase activity. Interestingly, the enzyme activity was increased approximately tenfold with 2.5 M NaCl or 3 M KCl. Furthermore, the optimal temperatures were 55°C with 2.5 M NaCl and 35°C without NaCl, respectively. This indicates that NaCl can improve enzyme thermostability. The K ( m ) and k (cat) values of CelB for CMC with 2.5 M NaCl were 3.18 mg mL(-1) and 26 s(-1), while the K ( m ) and k (cat) values of CelB without NaCl were 6.6 mg mL(-1) and 2.1 s(-1). Thus, this thermo-stable, salt and pH-tolerant cellulase is a promising candidate for industrial applications, and provides a new model to study salt effects on the structure of protein.

Significant progress has been made in isolating novel alkaline β-mannanases, however, there is a paucity of information concerning the structural basis for alkaline tolerance displayed by these β-mannanases. We report the catalytic domain structure of an industrially important β-mannanase from the alkaliphilic Bacillus sp. N16-5 (BSP165 MAN) at a resolution of 1.6 Å. This enzyme, classified into subfamily 8 in glycosyl hydrolase family 5 (GH5), has a pH optimum of enzymatic activity at pH 9.5 and folds into a classic (β/α)(8)-barrel. In order to gain insight into molecular features for alkaline adaptation, we compared BSP165 MAN with previously reported GH5 β-mannanases. It was revealed that BSP165 MAN and other subfamily 8 β-mannanases have significantly increased hydrophobic and Arg residues content and decreased polar residues, comparing to β-mannanases of subfamily 7 or 10 in GH5 which display optimum activities at lower pH. Further, extensive structural comparisons show alkaline β-mannanases possess a set of distinctive features. Position and length of some helices, strands and loops of the TIM barrel structures are changed, which contributes, to a certain degree, to the distinctly different shaped (β/α)(8)-barrels, thus affecting the catalytic environment of these enzymes. The number of negatively charged residues is increased on the molecular surface, and fewer polar residues are exposed to the solvent. Two amino acid substitutions in the vicinity of the acid/base catalyst were proposed to be possibly responsible for the variation in pH optimum of these homologous enzymes in subfamily 8 of GH5, identified by sequence homology analysis and pK(a) calculations of the active site residues. Mutational analysis has proved that Gln91 and Glu226 are important for BSP165 MAN to function at high pH. These findings are proposed to be possible factors implicated in the alkaline adaptation of GH5 β-mannanases and will help to further understanding of alkaline adaptation mechanism.

A xylanase gene (xyn10) from alkaliphilic Bacillus sp. N16-5 was cloned and expressed in Pichia pastoris. The deduced amino acid sequence has 85% identity with xylanase xyn10A from B. halodurans and contains two potential N-glycosylation sites. The glycosylated Xyn10 with MW 48 kDa can hydrolyze birchwood and oatspelt xylan. The enzyme had optimum activity at pH 7 and 70°C, with the specific activity of 92.5U/mg. The Xyn10 retained over 90% residual activity at 60°C for 30 min but lost all activity at 80°C over 15 min. Most tested ions showed no or slight inhibition effects on enzyme activity.