Novel compound heterozygous synonymous and missense variants in the MYO7A gene identified by next‐generation sequencing in a Chinese family with nonsyndromic hearing loss

BackgroundVariants in the MYO7A gene are increasingly identified among patients suffering from Usher syndrome type 1B (USH1B). However, such mutations are less commonly detected among patients suffering from nonsyndromic hearing loss (NSHL), including autosomal recessive deafness (DFNB2) and autosomal dominant deafness (DFNA11). This research attempts to clarify the genetic base of DFNB2 in a Chinese family and determine the pathogenicity of the identified mutations.MethodTargeted next‐generation sequencing (TGS) of 127 known deafness genes was performed for the 14‐year‐old proband. Then, Sanger sequencing was performed on the available family members. A minigene splicing assay was performed to verify the impact of the novel MYO7A synonymous variant. After performing targeted next‐generation sequencing (TGS) of 127 existing hearing loss‐related genes in a 14‐year‐old proband, Sanger sequencing was carried out on the available family members. Then, to confirm the influence of the novel MYO7A synonymous variants, a minigene splicing assay was performed.ResultsTwo heteroallelic mutants of MYO7A (NM_000260.3) were identified: a maternally inherited synonymous variant c.2904G > A (p.Glu968=) in exon 23 and a paternally inherited missense variant c.5994G > T (p.Trp1998Cys) in exon 44. The in vitro minigene expression indicated that c.2904G > A may result in skipping of exon 23 resulting in a truncated protein.ConclusionsWe reported a novel missense (c.5994G > T) and identified, for the first time, a novel pathogenic synonymous (c.2904G > A) variant within MYO7A in a patient with DFNB2. These findings enrich our understanding of the MYO7A variant spectrum of DFNB2 and can contribute to accurate genetic counseling and diagnosis of NSHL patients.     

GJB2 mutations in Iranian Azeri population with autosomal recessive nonsyndromic hearing loss (ARNSHL): First report of c.238 C>A mutation in Iran

ObjectiveAutosomal‐recessive nonsyndromic hearing loss (ARNSHL) is a heterogeneous genetic disorder. Mutations in the gap junction protein beta 2 (GJB2) gene, encoding connexin 26, are a significant cause of ARNSHL in different ethnic groups. This study aimed to identify the frequency and type of GJB2 mutations in the Iranian Azeri population.MethodsFifty unrelated families presenting ARNSHL in Ardabil Province, the northwest of Iran, were studied to determine the frequency and type of GJB2 mutations leading to ARNSHL. ARMS‐PCR screened all DNA samples to detect c.35delG; p. Gly12Val mutation. In addition, normal samples for c.35delG; p. Gly12Val were analyzed by direct sequencing for other GJB2 mutations.ResultOf the fifty families, 13 (26%) showed a GJB2 gene mutation, with c.35delG; p. Gly12Val mutation was the most prevalent one that occurred in eight (61.5%) out of the 13 families. Of the families, two were homozygous for c.358‐360delGAC; p. Glu120del mutation, and one was homozygous for c.290dupA; p. Tyr97Ter and c.299–300delAT; p. His100Arg mutations. Also, we detected a novel mutation, c.238C>A; p. Gln80lys, in one of the families.ConclusionOur findings are comparable to previous studies, indicating c.35d3lG; p. Gly12Val mutation in the GJB2 gene is the most common cause of GJB2‐related hearing loss in the Iranian Azeri population. Furthermore, our study highlights the significance of ARNSHL screening programs of live births based on local population data in Iran.     

Metagenomic next-generation sequencing for detection of pathogens in children with hematological diseases complicated with infection

ObjectiveInfection is one of the most common causes of death in children with hematological diseases. Here, we aim to investigate the value of metagenomic next-generation sequencing (mNGS) in the detection of causative pathogens in children with hematological diseases.MethodsIn this retrospective study, specimens from children with hematological diseases, who were admitted to Sun Yat-Sen University between June 2019 and September 2021, were collected for culture and mNGS.ResultsA total of 67 pediatric patients were enrolled, and 96 specimens were collected. The positive rate of mNGS was significantly higher than that of culture (57.2% vs 12.5%, P < 0.01). The concordance (90.9%, 10/11) between the positive results of the two methods was high. mNGS detected more cases with Pneumocystis jeroveci, Aspergillus flavus, viruses, and some rare pathogens than culture. Mixed infections were detected by mNGS in 16 cases. Clinical anti-infective treatment was adjusted according to the results of mNGS, the conditions of most patients improved.ConclusionCompared to culture, mNGS shows great advantages in diagnosing bacterial, fungal, viral, and mixed infections in children with hematologic diseases, positively impacting clinical care. mNGS can be used as a complement to culture for pathogen detection.     

一个携带线粒体tRNAHis12192G>A突变的耳聋家系研究

目的从临床、遗传和分子特征等方面对1个携带tRNAHis12192GA突变的非综合征型耳聋(NSHL)家系进行研究,探讨线粒体tRNAHis12192GA突变在NSHL中的作用。方法扩增572例NSHL患者和521例健康者GJB2、GJB3、GJB6基因编码区,以及线粒体12SrRNA和tRNA基因,对携带tRNAHis12192GA突变的1个家系进行GJB2、GJB3、GJB6、线粒体基因相关分析。结果在572例NSHL患者中发现1例线粒体tRNAHis12192GA突变携带者。该患者及其家族成员具有典型母系遗传特征,未检出GJB2、GJB3、GJB6基因致病突变。在患者家系成员中,母系成员13人,其中7人存在不同程度的听力损失。结论 tRNAHis12192GA突变可能是该NSHL家系成员罹患耳聋的主要分子基础。家系成员发病程度、发病年龄等表型存在一定的差异,可能与环境因素以核基因背景等有关。     

宏基因组学第二代测序技术对比传统实验室微生物培养在脓毒症病原学诊断中的优势

目的通过对宏基因组学第二代测序技术(mNGS)获得的病原体与实验室培养结果进行对比,了解mNGS在脓毒症病原学诊断中的优势及其临床指导意义。方法将入选的脓毒症患者的标本(肺泡灌洗液、痰液、血液、脑脊液、胸水、腹水、分泌物等)同时送检mNGS和实验室细菌培养,对结果进行对比分析,评价mNGS在脓毒症病原学诊断方面的临床价值。结果mNGS的阳性率为78.9%;细菌培养的阳性率为40.4%(P<0.05)。通过mNGS共检出致病病原体57种,其中细菌31种,真菌16种,病毒7种,非典型病原体3种;细菌培养共检出病原体24种,其中细菌18种,真菌6种。以培养结果为金标准,mNGS的敏感度为76.2%,特异度为29.8%,阳性预测值为42.3%,阴性预测值为64.8%。根据病原学结果的抗生素调整将患者分为三组:按mNGS调整为mNGS组、经验性调整为经验组、按培养结果调整为传统培养组。mNGS组ICU住院时间更短(P<0.05),培养组降钙素原下降更明显(P<0.05)。结论mNGS在感染性疾病病原体的诊断方面较传统微生物培养时间更短,阳性率更高,在少见病原体、罕见病原体诊断方面有显著优势,可缩短患者ICU住院时间。     

Identification of novel variants in ten patients with Hermansky-Pudlak syndrome by high-throughput sequencing

Background: Hermansky-Pudlak syndrome (HPS) is a rare inherited platelet disorder characterized by bleeding diathesis, oculocutaneous albinism (OCA) and a myriad of often-serious clinical complications.Methods: We established the clinical and laboratory phenotype and genotype of six unrelated pedigrees comprising ten patients with clinical suspicion of HPS; including platelet aggregation, flow cytometry, platelet dense granule content, electron microscopy and high-throughput sequencing (HTS).Results: The clinical presentation showed significant heterogeneity and no clear phenotype-genotype correlations. HTS revealed two known and three novel disease-causing variants. The Spanish patients carried a homozygous p.Pro685Leufs17* deletion (n = 2) in HPS4, or the novel p.Arg822* homozygous variant (n = 1) in HPS3. In the case of two Turkish sisters, a novel missense homozygous HPS4 variant (p.Leu91Pro) was found. In two Portuguese families, genetic studies confirmed a previously reported nonsense variant (p.Gln103*) in DTNBP1 in three patients and a novel duplication (p.Leu22Argfs*33) in HPS6 in two unrelated patients.Conclusions: Our findings expand the mutational spectrum of HPS, which may help in investigating phenotype-genotype relationships and assist genetic counselling for affected individuals. This approach is a proof of principle that HTS can be considered and used in the first-line diagnosis of patients with biological and clinical manifestations suggestive of HPS.

Key messages

• We established the relationships between the clinical and laboratory phenotype and genotype of six unrelated pedigrees comprising ten patients with clinical suspicion of HPS.
• Molecular analysis is useful in confirming the diagnosis and may offer some prognostic information that will aid in optimizing monitoring and surveillance for early detection of end-organ damage.
• This approach is a proof of principle that HTS can be considered and used in the first-line diagnosis of patients with biological and clinical manifestations suggestive of HPS.

     

Identification of germline pathogenic variants in DNA damage repair genes by a next-generation sequencing multigene panel in BRCAX patients

BackgroundApproximately 5–10% of breast carcinomas have been related to hereditary conditions and are attributable to pathogenic variants in the BRCA1 and BRCA2 genes, which is referred to as hereditary breast and ovarian cancer (HBOC) syndrome. The inclusion of additional genes that can be related to HBOC syndrome is under intense evaluation due to the high proportion of patients with HBOC criteria who do not present pathogenic mutations in BRCA genes, named BRCAX, despite having high clinical suspicion of hereditary cancer. The main aim is to identify new potentially pathogenic gene variants that may contribute to HBOC to improve the efficiency of routine diagnostic tests in this hereditary condition.MethodsA retrospective cohort of 77 HBOC BRCAX patients was analyzed by next-generation sequencing using a targeted multigene panel composed of 25 genes related to hereditary cancer and deficiencies in DNA repair pathways.ResultsWe found 9 variants in 7 different genes, which were confirmed by automated sequencing. Six variants were classified as pathogenic or likely pathogenic. Three of them were located in the PALB2 gene, one in the BRIP1 gene, one in the BARD1 gene and 1 in the RAD50 gene. In addition, three variants of uncertain significance (VUS) were detected in the TP53, CHEK2, and CDH1 genes.ConclusionsWe identified that 8% of BRCAX patients were carriers of pathogenic variants in genes other than BRCA1 and BRCA2. Therefore, wide gene panels, including clinically actionable genes, should be routinely used in the screening of HBOC in our population. We observed differences from other studies in the prevalence of mutated genes, most likely due to differences in the selection criteria of the probands and in the population analyzed. The high incidence of deleterious variant detection in PALB2 supports its significant role in breast cancer susceptibility and reinforces its inclusion in the HBOC genetic diagnostic process.     

第二代测序技术检测1例假肥大型肌营养不良家系Dystrophin基因突变

目的探讨第二代测序(NGS)技术检测假肥大型肌营养不良患者的可行性。方法用NGS技术检测1例假肥大型肌营养不良(DMD)患者,并用Sanger测序技术对患者基因型进行验证,同时检测家系其他成员基因型。结果 NGS技术结果表明,该患者DMD基因编码区第55号外显子存在1个移码突变c.8087delT(p.Leu2696ArgfsX30),为致病性突变;同时该患者还存在3个错义突变p.Arg2937Gln、p.Arg1745His和p.Asp882Gly,均为已知多态性变异;Sanger测序技术进一步证实了该点突变的存在,同时发现患者母亲为该突变的携带者,患者父亲和妹妹未见该突变。结论 NGS技术可用于DMD基因突变检测,为DMD临床遗传咨询和治疗提供了依据。     

脂联素和Hs-CRP在突发性耳聋中的临床意义

目的探讨突发性耳聋患者血清脂联素和血清高敏c一反应蛋白（Hs-CRP）的含量变化,及其在突发性耳聋发生过程中的作用和意义。方法采用酶联免疫（ELISA）法检测血清脂联素含量,采用免疫比浊法对血清Hs—CRP进行测定;检测102例突发性耳聋患者、35例其他疾病对照组和30例正常健康对照组的血清脂联素和Hs-CRP含量,并比较治疗前后血清脂联素和Hs—CRP的测定结果。结果突发性耳聋患者治疗前血清Hs-CRP含量较两对照组显著升高（P〈0．01）;治疗后恢复组患者Hs-CRP含量明显降低。突发性耳聋患者治疗前血清脂联素含量较两对照组显著降低（P〈0．01）;治疗后恢复组患者脂联素含量明显升高。突发性耳聋患者血清脂联素和Hs—CRP含量之间呈负相关（r=-0．63,P〈0．01）。结论血清脂联素和Hs—CRP水平的变化与突发性耳聋病情严重程度密切相关。     

Identification of a novel truncating mutation in PALB2 gene by a multigene sequencing panel for mutational screening of breast cancer risk‐associated and related genes

Background

Breast cancer (BC) is the most common neoplasm in women, with 5%‐10% patients showing a familial predisposition, where germline mutations in BRCA1/BRCA2 genes are found in –20% of cases. Next‐generation sequencing (NGS) is among the best available options for genetic screening, providing several benefits that include enhanced sensitivity and unbiased mutation detection. PALB2 (partner and localizer of BRCA2) is a cancer predisposing gene recently described that encodes a protein partner of BRCA2 involved in DNA double‐strand break repair and cell cycle control. The DNA damage response represents a key cellular event, targeted by innovative anticancer therapies, including those based on poly (ADP‐ribose) polymerase (PARP) inhibitors targeting PARP1 and PARP2 enzymes, activated by DNA damage and involved in single‐strand break and base excision repair.

Methods

Genomic DNA was isolated from 34 patient samples and four BC cell lines, as controls, and 27 breast cancer predisposing genes belonging to the BRCA1/BRCA2 and PARP pathways were sequenced by NGS.

Results

The panel described here allowed identification of several sequence variations in most investigated genes, among which we found a novel truncating mutation in PALB2.

Conclusions

The NGS‐based strategy designed here for molecular analysis of a customized panel of BC predisposing and related genes was found to perform effectively, providing a comprehensive exploration of all genomic sequences of the investigated genes. It is thus useful for BC molecular diagnosis, in particular for familiar cases where alterations in routinely investigated genes, such as BRCAs, result to be absent.

     

Mutational spectrum of the phenylalanine hydroxylase gene in patients with phenylketonuria in the central region of China

Phenylketonuria (PKU, OMIM 261600) caused by phenylalanine hydroxylase (PAH) deficiency is an autosomal recessive disease that is characterized by abnormalities of phenylalanine metabolism. In this study, a total of 77 patients, originating from the central region of China and who were diagnosed with PAH deficiency at the third affiliated hospital of Zhengzhou University, were enrolled in this study. The 13 exons and 12 flanking introns of the PAH gene were analyzed by Sanger sequencing and next generation sequencing. The sequencing data were aligned to the hg19, PAHvdb and HGMD databases to characterize the genotypes of PKU patients, and genotype–phenotype correlations and BH4 responsiveness predictions were performed using BIOPKUdb. In total, 149 alleles were characterized among the 154 PKU alleles. These mutations were located in exons 2–13, and intron 12 of the PAH gene, with a relative frequency of ≥5%, for EX6-96A>G, p.R241C, p.R243Q, p.V399V and p.R53H. Additionally, a novel variant, p.D84G, was identified. The genotype correlated with clinical symptoms in 33.3–100% of the cases, depending on the disease severity, and BH4 responsiveness predictions show that only five patients with MHP-PKU and one patient with Mild-PKU were predicted to be BH4 responsive. In conclusion, we have characterized the mutational spectrum of PAH in the central region of China and have identified a novel mutation. The hotspot mutation information might be useful for screening, diagnosis and treatment of PKU.     

Identification of a novel EXT2 frameshift mutation in a family with hereditary multiple exostoses by whole‐exome sequencing

BackgroundHereditary multiple exostoses (HME), also referred to as multiple osteochondromas, is an autosomal dominant skeletal disease characterized by the development of multiple overgrown benign bony tumors capped by cartilage and is associated with bone deformity, joint limitation, and short stature. Mutations in exostosin glycosyltransferase (EXT)1 and EXT2 genes, which are located on chromosomes 8q24.1 and 11p13, contribute to the pathogenesis of HME.MethodsIn the present study, a genetic analysis of a four‐generation Chinese family with HME was conducted using whole‐exome sequencing (WES), followed by validation using Sanger sequencing.ResultsA novel heterozygous frameshift mutation in exon 5 of EXT2 (c.944dupT, p.Leu316fs) was identified in all affected individuals but was not detected in any unaffected individuals. This mutation results in a frameshift that introduces a premature termination codon at position 318 (p.Leu316fs) with the ability to produce a truncated EXT2 protein that lacks the last 433 amino acids at its C‐terminal to indicate a defective exostosin domain and the absence of the glycosyltransferase family 64 domain, or to lead to the degradation of mRNAs by nonsense‐mediated mRNA decay, which is critical for the function of EXT2.ConclusionOur results indicate that WES is effective in extending the EXT mutational spectra and is advantageous for genetic counseling and the subsequent prenatal diagnosis.