BackgroundDuchenne Muscular Dystrophy (DMD) is the most common muscle disease in children, and there are no effective therapies for DMD or Becker Muscular Dystrophy (BMD). Currently, targeted gene therapy treatments have emerged. As a result, genetic diagnosis is the basis of treatment. In addition, genetic and prenatal diagnosis significantly reduces their incidence rates. This study combines the application of multiplex ligation-dependent probe amplification technology (MLPA) and “next-generation” sequencing technology (NGS) as the most economical and efficient method of diagnosis. Therefore, in the diagnosis of DMD/BMD, patients’ MLPA data are first used to detect DMD gene deletions or duplications, and NGS and Sanger sequencing are then applied to exclude MLPA-negative samples. Meanwhile, polymerase chain reaction (PCR) is used to detect single exon deletions to exclude false-positives in MLPA caused by point mutations.MethodsIn this study, we recruited 1051 proband families of DMD from 2016 to 2018 and had access to information that could identify individual participants during or after data collection. Patients who were diagnosed with DMD were first tested by MLPA. MLPA results with single exon deletions were validated with PCR amplification and Sanger sequencing. The negative results of MLPA were further analysed with NGS and validated by Sanger sequencing. For novel missense mutations, phenotype-genotype correlations were analysed using PolyPhen2 and mutation taster. All methods were performed in accordance with the relevant guidelines and regulations.ResultsDMD mutations were identified in 1029 families (97.91%, 1029/1051). Large deletions, duplications, and small mutations accounted for 70.41% (740/1051), 8.28% (87/1051), and 19.12% (201/1051) of all cases, respectively. There were 205 small mutation types, 53 of which were novel. The rate of de novo mutations was 39.45% (187/474) and was higher in large duplications (49.53%, 157/317). Among 68 asymptomatic patients (< 3 years old) with unexplained persistent hyperCKaemia upon conventional physical examination, 63 were diagnosed as DMD/BMD according to genetic diagnosis.ConclusionOur results expand the spectrum of DMD mutations, which could contribute to the treatment of DMD/BMD and provide an effective diagnosis method. Thus, the combination of MLPA, NGS and Sanger sequencing is of great significance for family analysis, gene diagnosis and gene therapy.

BackgroundBartter syndrome (BS) is a rare autosomal recessive disorder of salt reabsorption at the thick ascending limb of the Henle loop, characterized by hypokalemia, salt loss, metabolic alkalosis, hyperreninemic hyperaldosteronism with normal blood pressure. BS type III, often known as classic BS (CBS), is caused by loss-of-function mutations in CLCNKB (chloride voltage-gated channel Kb) encoding basolateral ClC-Kb.Case presentationWe reported a 15-year-old CBS patient with a compound heterozygous mutation of CLCNKB gene. She first presented with vomiting, hypokalemic metabolic alkalosis at the age of 4 months, and was clinically diagnosed as CBS. Indomethacin, spironolactone and oral potassium were started from then. During follow-up, the serum electrolyte levels were generally normal, but the patient showed failure to thrive and growth hormone (GH) deficiency was diagnosed. The recombinant human GH therapy was performed, and the growth velocity was improved. When she was 14, severe proteinuria and chronic kidney disease (CKD) were developed. Renal biopsy showed focal segmental glomerulosclerosis (FSGS) with juxtaglomerular apparatus cell hyperplasia, and genetic testing revealed a point deletion of c.1696delG (p. Glu566fs) and a fragment deletion of exon 2–3 deletions in CLCNKB gene. Apart from the CBS, ostium secundum atrial septal defect (ASD) was diagnosed by echocardiography.ConclusionsThis is the first report of this compound heterozygous of CLCNKB gene in BS Children. Our findings contribute to a growing list of CLCNKB mutations associated with CBS. Some recessive mutations can induce CBS in combination with other mutations.

BackgroundOculocutaneous 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 presentationHere, 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.ConclusionsThis 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.

BackgroundSeveral studies have been performed to investigate the associations between interleukin (IL)-8 rs4073 polymorphism and acute pancreatitis (AP), but the results are inconclusive. We conducted this cumulative meta-analysis for a precise estimate of the relationship between IL-8 rs4073 polymorphism and acute pancreatitis.MethodsWe searched the electronic databases for relevant studies. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the strength of the associations. For a better presentation of how the pooled ORs changed as updated evidence accumulated, we used forest plots from a cumulative meta-analysis method.ResultsTen studies involving 1646 AP patients and 1816 controls were finally included in this meta-analysis. Cumulative meta-analyses indicated there is a consistent trend toward association after the initial discovery. Under the allelic, dominant, recessive and homozygous models, the pooled ORs were 1.265 (1.147–1.395, p < 0.001), 1.304 (1.127–1.508, p < 0.001), 1.431 (1.203–1.702, p < 0.001), and 1.634 (1.334–2.001, p < 0.001), respectively.ConclusionsThis meta-analysis demonstrated a suggestive result that people who carried the risk A allele of the IL-8 rs4073 polymorphism may be more sensitive to acute pancreatitis.

BackgroundDiamond-Blackfan anemia (DBA), a congenital pure red cell aplasia (PRCA), is characterized by normochromic macrocytic anemia, reticulocytopenia, and nearly absent erythroid progenitors in the bone marrow. DBA10, a subset of DBA, is an autosomal dominant disease caused by a mutation in RPS26. So far, there are 30 disease-causing variants in RPS26 being reported, however, only three of them are small insert mutations.Case presentationHere we report a three-month Chinese boy who presents with anemia from postnatal day 2. He was suspected to have Diamond-Blackfan anemia, according to the clinical result. Thus, whole-exome sequencing was performed for precise diagnosis.ConclusionHere, a novel insert mutation c.96dupG in RPS26 was identified by whole-exome sequencing, which caused neonatal DBA in a Chinese boy. This is the first case report of a Chinese DBA10 patient who carries a small insertion in the RPS26 gene. These findings expand the mutation diversity of RPS26 and demonstrate the clinical presentations of the Chinese DBA10 patient.

BackgroundFocal segmental glomerulosclerosis (FSGS) is still one of the common causes of refractory nephrotic syndrome. Nephrin, encoded by podocyte-specific NPHS1 gene, participated in the pathogenesis of FSGS. The sites of NPHS1 mutations in FSGS is not clarified very well. In this study, we investigated the specific mutations of NPHS1 gene in Chinese patients with sporadic FSGS.MethodsA total of 309 patients with sporadic FSGS were collected and screened for NPHS1 mutations by second-generation sequencing. The variants were compared with those extracted from 2504 healthy controls in the 1000 Genomes Project. The possible pathogenic roles of missense variants were predicted by three different software. We also compared these candidate causal mutations with those summarized from the previous studies.ResultsThirty-two genetic mutations of NPHS1 gene were identified in FSGS patients, including 12 synonymous mutations, 17 missense mutations, 1 splicing mutation, and 2 intron mutations, of which c.G3315A (p.S1105S) was the most common variant (261/309). A novel missense mutation c.G2638 T (p.V880F) and a novel splicing mutation 35830957 C > T were identified in FSGS patients. The frequencies of the four synonymous mutations (c.C294T [p.I98I], c.C2223T [p.T741 T], c.C2289T [p.V763 V], c.G3315A [p.S1105S]) were much higher in FSGS patients than in controls. The frequencies of the four missense mutations (c.G349A [p.E117K], c.G1339A [p.E447K], c.G1802C [p.G601A], c.C2398T [p.R800C]) were much higher and one (c.A3230G [p.N1077S]) was lower in FSGS patients than in controls. Five missense mutations, c.C616A (p.P206T), c.G1802C (p.G601A), c.C2309T (p.P770L), c.G2869C (p.V957 L), and c.C3274T (p.R1092C), were predicted to be pathogenic mutations by software analysis.ConclusionsNPHS1 gene mutations were quite common in sporadic FSGS patients. We strongly recommend mutation analysis of the NPHS1 gene in the clinical management of FSGS patients.

BackgroundX-linked Adrenal Hypoplasia Congenita (AHC) is a rare cause of primary adrenal insufficiency due to mutations in the NR0B1 gene, causing a loss of function of the nuclear receptor protein DAX-1. Adrenal insufficiency usually appears in the first 2 months of life, but can sometimes emerge during childhood. Hypogonadotropic Hypogonadism is often associated later in life and patients may develop azoospermia. We describe an unusual onset of AHC started with isolated hypoaldosteronism as first and only sign of the disease.Case presentationA 18-days-old newborn presented with failure to thrive and feeding difficulties. Blood tests showed severe hyponatremia, hyperkalemia and hypochloremia. Renin was found over the measurable range and aldosterone was low whereas cortisol level was normal with a slightly increased ACTH. In the suspicion of Primary Hypoaldosteronism, correction of plasmatic electrolytes and replacement therapy with Fludrocortisone were promptly started. The subsequent evidence of low plasmatic and urinary cortisol and increased ACTH required the start of Hydrocortisone replacement therapy and it defined a clinical picture of adrenal insufficiency. Genetic analysis demonstrated a novel mutation in the DAX-1 gene leading to the diagnosis of AHC.ConclusionsAHC onset may involve the aldosterone production itself, miming an isolated defect of aldosterone synthesis. NR0B1/DAX-1 mutations should be considered in male infants presenting with isolated hypoaldosteronism as first sign of adrenal insufficiency.

BackgroundGenome wide association study (GWAS) has become the major means to screen for the genetic variants associated with risk and prognosis of different diseases. A recent GWAS has discovered three novel intronic single nucleotide polymorphisms in genes LRFN2 (rs2494938), DNAH11 (rs2285947) and PLCXD2 (rs2399395) that are associated with altered risk of esophageal squamous cell carcinoma (ESCC) among Han Chinese populations. However, the prognostic significance of these variations in ESCC remains unclear.MethodsTo investigate the association of three novel single nucleotide polymorphisms (rs2494938, rs2285947, rs2399395) with the prognosis of ESCC patients, we recruited 287 ESCC patients treated with surgical resection and evaluated the potential significance of the three polymorphisms through Kaplan-Meier survival analysis, log-rank test, and Cox proportional hazards regression models.ResultsThe ESCC patients carrying genotype AA at rs2494938 had worse survival and genotype GG at 2285947 had better prognosis (Log-rank P = 0.003 and Log-rank P = 0.037, respectively). In addition, rs2494938 at 6p21.1 was independently associated with overall survival of ESCC patients in recessive model [AA vs. GG/GA, HR = 3.12, 95% CI = 1.43–6.83, P = 0.004], rs2285947 at 7p15.3 was independently associated with overall survival of ESCC patients in both dominant model [AA/GA vs. GG, HR = 1.59, 95% CI = 1.02–2.49, P = 0.042] and additive model [AA vs. GA vs. GG, HR = 1.45, 95% CI = 1.05–2.01, P = 0.025].ConclusionsThis study demonstrated that the polymorphisms rs2494938 at 6p21.1 and rs2285947 at 7p15.3 may serve as independent prognostic biomarkers for ESCC, implying the potential biological role of their related genes (LRFN2 and DNAH11) in the process of ESCC development.

BackgroundMYO15A variants are responsible for human non-syndromic autosomal recessive deafness (DFNB3). The majority of MYO15A variants are associated with a congenital severe-to-profound hearing loss phenotype, except for MYO15A variants in exon 2, which cause a milder auditory phenotype, suggesting a genotype-phenotype correlation of MYO15A. However, MYO15A variants not in exon 2 related to a milder phenotype have also been reported, indicating that the genotype-phenotype correlation of MYO15A is complicated. This study aimed to provide more cases of MYO15A variation with diverse phenotypes to analyse this complex correlation.MethodsFifteen Chinese autosomal recessive non-syndromic hearing loss (ARNSHL) individuals with MYO15A variants (8 males and 7 females) from 14 unrelated families, identified by targeted gene capture of 127 known candidate deafness genes, were recruited. Additionally, we conducted a review of the literature to further analyses all reported MYO15A genotype-phenotype relationships worldwide.ResultsWe identified 16 novel variants and 12 reported pathogenic MYO15A variants in 15 patients, two of which presented with a milder phenotype. Interestingly, one of these cases carried two reported pathogenic variants in exon 2, while the other carried two novel variants not in exon 2. Based on our literature review, MYO15A genotype-phenotype correlation analysis showed that almost all domains were reported to be correlated with a milder phenotype. However, variants in the N-terminal domain were more likely to cause a milder phenotype. Using next-generation sequencing (NGS), we also found that the number of known MYO15A variants with milder phenotypes in Southeast Asia has increased in recent years.ConclusionOur work extended the MYO15A variant spectrum, enriched our knowledge of auditory phenotypes, and tried to explore the genotype-phenotype correlation in different populations in order to investigate the cause of the complex MYO15A genotype-phenotype correlation.

BackgroundHyperprolactinemia (HPRL) is a classical side effect of antipsychotic drugs primarily attributed to blockade of dopamine D2 receptors (DRD2s) on the membranes of lactotroph cells within the pituitary gland. Certain antipsychotic drugs, e.g. risperidone, are more likely to induce HPRL because of relative accumulation within the adenohypophysis. Nevertheless, due to competition for pituitary DRD2s by high dopamine levels may limit antipsychotic-induced HPRL. Moreover, the activity of prolactin-producing lactotrophs also depends on other hormones which are regulated by the extra-pituitary activity of dopamine receptors, dopamine transporters, enzymes of neurotransmitter metabolism and other factors. Polymorphic variants in the genes coding for these receptors and proteins can have functional significance and influence on the development of hyperprolactinemia.MethodsA set of 41 SNPs of genes for dopamine receptors DRD1, DRD2, DRD3, DRD4, the dopamine transporter SLC6A3 and dopamine catabolizing enzymes MAOA and MAOB was investigated in a population of 446 Caucasians (221 males/225 females) with a clinical diagnosis of schizophrenia (according to ICD-10: F20) with and without HPRL who were treated with classical and/or atypical antipsychotic drugs. Additive genetic model was tested and the analysis was carried out in the total group and in subgroup stratified by the use of risperidone/paliperidone.ResultsOne statistically significant association between polymorphic variant rs1799836 of MAOB gene and HPRL in men was found in the total group. Furthermore, the rs40184 and rs3863145 variants in SLC6A3 gene appeared to be associated with HPRL in the subgroup of patients using the risperidone/paliperidone, but not with HPRL induced by other antipsychotic drugs.ConclusionsOur results indicate that genetic variants of MAOB and SLC6A3 may have consequences on the modulation of prolactin secretion. A further search for genetic markers associated with the development of antipsychotic-related hyperprolactinemia in schizophrenic patients is needed.