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Inherited retinal diseases (IRDs) are a group of rare genetic eye conditions that cause blindness. Despite progress in identifying genes associated with IRDs, improvements are necessary for classifying rare autosomal dominant (AD) disorders. AD diseases are highly heterogenous, with causal variants being restricted to specific amino acid changes within certain protein domains, making AD conditions difficult to classify. Here, we aim to determine the top-performing in-silico tools for predicting the pathogenicity of AD IRD variants. We annotated variants from ClinVar and benchmarked 39 variant classifier tools on IRD genes, split by inheritance pattern. Using area-under-the-curve (AUC) analysis, we determined the top-performing tools and defined thresholds for variant pathogenicity. Top-performing tools were assessed using genome sequencing on a cohort of participants with IRDs of unknown etiology. MutScore achieved the highest accuracy within AD genes, yielding an AUC of 0.969. When filtering for AD gain-of-function and dominant negative variants, BayesDel had the highest accuracy with an AUC of 0.997. Five participants with variants in NR2E3, RHO, GUCA1A, and GUCY2D were confirmed to have dominantly inherited disease based on pedigree, phenotype, and segregation analysis. We identified two uncharacterized variants in GUCA1A (c.428T>A, p.Ile143Thr) and RHO (c.631C>G, p.His211Asp) in three participants. Our findings support using a multi-classifier approach comprised of new missense classifier tools to identify pathogenic variants in participants with AD IRDs. Our results provide a foundation for improved genetic diagnosis for people with IRDs., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Mutations in the MYO6 gene have been associated with both autosomal dominant non-syndromic hearing loss (ADNSHL) and autosomal recessive non-syndromic hearing loss (ARNSHL), with a cumulative identification of 125 pathogenic variants. To investigate the underlying genetic factor within a Chinese family affected with heriditary hearing loss, prompted the utilization of high-throughput sequencing. Method: A detailed clinical investigation was performed. Genetic testing was performed by using target panel sequencing, and Sanger sequencing. Targeted sequencing identified the variants and Sanger sequencing was employed to validate segregation of the identified variants within family. Additionally, bioinformatics analysis was performed to strengthen our findings. Results: Clinical investigation revealed the family members were affected by progressive and sensorineural hearing loss with an onset around 8-10 years old. Furthermore, genetic testing identified novel MYO6 variants, c.[2377T>G; 2382G>T] p.[Trp793Gly; Lys794Asn], positioned in a cis pattern, as plausible pathogenic contributors to early-onset hearing loss characterized by a severe and progressive course. Moreover, bioinformatics analysis showd disruptin in hydrogen bonding of mutant amino acids with interactive amino acids. Conclusion: Our research uncovered a relationship between mutations in the MYO6 gene and non-syndromic hearing loss. We identified two variants, c.[2377T>G; 2382G>T] p.[Trp793Gly; Lys794Asn] in MYO6 as strong candidates responsible for the observed progressive hereditary hearing loss. This study not only adds to our knowledge about hearing problems related to MYO6 but also reveals the presence of monogenic compound heterozygosity. Our study will provide a new sight for genetic diagnosis in such patients and their management for future use., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ji, Zhang, Hussain, Aftab, Yu and Xiao.)

Neuromuscular disorders encompass a broad range of phenotypes and genetic causes. We investigated a consanguineous family in which multiple patients had a neuromuscular disorder characterized by a waddling gait, limb deformities, muscular weakness and facial palsy. Exome sequencing was completed on the DNA of three of the four patients. We identified a novel missense variant in DCAF13, ENST00000612750.5, NM_015420.7, c.907 G > A;p.(Asp303Asn), ENST00000616836.4, NM_015420.6, c.1363 G > A:p.(Asp455Asn) (rs1209794872) segregating with this phenotype; being homozygous in all four affected patients and heterozygous in the unaffected individuals. The variant was extremely rare in the public databases (gnomAD allele frequency 0.000007081); was absent from the DNA of 300 ethnically matched controls and affected an amino acid which has been conserved across 1-2 billion years of evolution in eukaryotes. DCAF13 contains three WD40 domains and is hypothesized to have roles in both rRNA processing and in ubiquitination of proteins. Analysis of DCAF13 with the p.(Asp455Asn) variant predicted that the amino acid change is deleterious and affects a β-hairpin turn, within a WD40 domain of the protein which may decrease protein stability. Previously, a heterozygous variant of DCAF13 NM_015420.6, c.20 G > C:p.(Trp7Ser) with or without a heterozygous missense variant in CCN3, was suggested to cause inherited cortical myoclonic tremor with epilepsy. In addition, a heterozygous DCAF13 variant has been associated with autism spectrum disorder. Our study indicates a potential role of biallelic DCAF13 variants in neuromuscular disorders. Screening of additional patients with similar phenotype may broaden the allelic and phenotypic spectrum due to DCAF13 variants., (© 2023. The Author(s), under exclusive licence to European Society of Human Genetics.)

Inherited retinal diseases (IRDs) are a diverse set of visual disorders that collectively represent a major cause of early-onset blindness. With the reduction in sequencing costs in recent years, whole-genome sequencing (WGS) is being used more frequently, particularly when targeted gene panels and whole-exome sequencing (WES) fail to detect pathogenic mutations in patients. In this study, we performed mutation screens using WGS for a cohort of 311 IRD patients whose mutations were undetermined. A total of nine putative pathogenic mutations in six IRD patients were identified, including six novel mutations. Among them, four were deep intronic mutations that affected mRNA splicing, while the other five affected protein-coding sequences. Our results suggested that the rate of resolution of unsolved cases via targeted gene panels and WES can be further enhanced with WGS; however, the overall improvement may be limited.

Background: Focal and segmental glomerulosclerosis (FSGS) is a histological pathology that characterizes a wide spectrum of diseases. Many genes associated with FSGS have been studied previously, but there are still some FSGS families reported in the literature without the identification of known gene mutations. The aim of this study was to investigate the new genetic cause of adult-onset FSGS., Methods: This study included 40 FSGS families, 77 sporadic FSGS cases, 157 non-FSGS chronic kidney disease (CKD) families and 195 healthy controls for analyses. Whole-exome sequencing (WES) and Sanger sequencing were performed on probands and family members of all recruited families and sporadic FSGS cases., Results: Using WES, we have identified a novel heterozygous missense variant (c.T1655C:p.V552A) in exportin 5 gene (XPO5) in two families (FS-133 and CKD-05) affected with FSGS and CKD. Sanger sequencing has confirmed the co-segregation of this identified variant in an autosomal dominant pattern within two families, while this variant was absent in healthy controls. Furthermore, the identified mutation was absent in 195 ethnically matched healthy controls by Sanger sequencing. Subsequently, in silico analysis demonstrated that the identified variant was highly conservative in evolution and likely to be pathogenic., Conclusions: Our study reports an adult-onset autosomal dominant inheritance of the XPO5 variant in familial FSGS for the first time. Our study expanded the understanding of the genotypic, phenotypic and ethnical spectrum of mutation in this gene., (© 2022. The Author(s).)

Background: The XRCC3 p.Thr241Met (rs861539) polymorphism has been extensively studied for its association with glioma risk, but results remain conflicting. Therefore, we performed a systematic review and meta-analysis to resolve this inconsistency., Methods: Studies published up to June 10, 2022, were searched in PubMed, Web of Science, Scopus, VIP, Wanfang, and China National Knowledge Infrastructure databases and screened for eligibility. Then, the combined odds ratio (OR) of the included studies was estimated based on five genetic models, i.e., homozygous (Met/Met vs. Thr/Thr), heterozygous (Thr/Met vs. Thr/Thr), dominant (Thr/Met + Met/Met vs. Thr/Thr), recessive (Met/Met vs. Thr/Thr + Thr/Met) and allele (Met vs. Thr). The study protocol was preregistered at PROSPERO (registration number: CRD42021235704)., Results: Overall, our meta-analysis of 14 eligible studies involving 12,905 subjects showed that the p.Thr241Met polymorphism was significantly associated with increased glioma risk in both homozygous and recessive models (homozygous, OR = 1.381, 95% CI = 1.081-1.764, P = 0.010; recessive, OR = 1.305, 95% CI = 1.140-1.493, P<0.001). Subgroup analyses by ethnicity also revealed a statistically significant association under the two aforementioned genetic models, but only in the Asian population and not in Caucasians (P>0.05)., Conclusion: We demonstrated that the XRCC3 p.Thr241Met polymorphism is associated with an increased risk of glioma only in the homozygous and recessive models., Competing Interests: The authors declare no conflicts of interest.

Background: This study sought to investigate incidence and risk factors for acute kidney injury (AKI) in hospitalized COVID-19., Methods: In this retrospective study, we enrolled 823 COVID-19 patients with at least two evaluations of renal function during hospitalization from four hospitals in Wuhan, China between February 2020 and April 2020. Clinical and laboratory parameters at the time of admission and follow-up data were recorded. Systemic renal tubular dysfunction was evaluated via 24-h urine collections in a subgroup of 55 patients., Results: In total, 823 patients were enrolled (50.5% male) with a mean age of 60.9 ± 14.9 years. AKI occurred in 38 (40.9%) ICU cases but only 6 (0.8%) non-ICU cases. Using forward stepwise Cox regression analysis, we found eight independent risk factors for AKI including decreased platelet level, lower albumin level, lower phosphorus level, higher level of lactate dehydrogenase (LDH), procalcitonin, C-reactive protein (CRP), urea, and prothrombin time (PT) on admission. For every 0.1 mmol/L decreases in serum phosphorus level, patients had a 1.34-fold (95% CI 1.14-1.58) increased risk of AKI. Patients with hypophosphatemia were likely to be older and with lower lymphocyte count, lower serum albumin level, lower uric acid, higher LDH, and higher CRP. Furthermore, serum phosphorus level was positively correlated with phosphate tubular maximum per volume of filtrate (TmP/GFR) (Pearson r  = 0.66, p  < .001) in subgroup analysis, indicating renal phosphate loss via proximal renal tubular dysfunction., Conclusion: The AKI incidence was very low in non-ICU patients as compared to ICU patients. Hypophosphatemia is an independent risk factor for AKI in patients hospitalized for COVID-19 infection.

Introduction: Acute kidney injury (AKI) in coronavirus disease 2019 (COVID-19) patients is associated with poor prognosis. Early prediction and intervention of AKI are vital for improving clinical outcome of COVID-19 patients. As lack of tools for early AKI detection in COVID-19 patients, this study aimed to validate the USCD-Mayo risk score in predicting hospital-acquired AKI in an extended multi-center COVID-19 cohort., Methods: Five hundred seventy-two COVID-19 patients from Wuhan Tongji Hospital Guanggu Branch, Wuhan Leishenshan Hospital, and Wuhan No. Ninth Hospital was enrolled for this study. Patients who developed AKI or reached an outcome of recovery or death during the study period were included. Predictors were evaluated according to data extracted from medical records., Results: Of all patients, a total of 44 (8%) developed AKI. The UCSD-Mayo risk score achieved excellent discrimination in predicting AKI with the C-statistic of 0.88 (95%CI: 0.84-0.91). Next, we determined the UCSD-Mayo risk score had good overall performance (Nagelkerke R 2  = 0.32) and calibration in our cohort. Further analysis showed that the UCSD-Mayo risk score performed well in subgroups defined by gender, age, and several chronic comorbidities. However, the discrimination of the UCSD-Mayo risk score in ICU patients and patients with mechanical ventilation was not good which might be resulted from different risk factors of these patients., Conclusions: We validated the performance of UCSD-Mayo risk score in predicting hospital-acquired AKI in COVID-19 patients was excellent except for patients from ICU or patients with mechanical ventilation.

We aimed to validate three IgAN risk models proposed by an international collaborative study and another CKD risk model generated by an extended CKD cohort with our multicenter Chinese IgAN cohort. Biopsy-proven IgAN patients with an eGFR ≥15 ml/min/1.73 m 2 at baseline and a minimum follow-up of 6 months were enrolled. The primary outcomes were a composite outcome (50% decline in eGFR or ESRD) and ESRD. The performance of those models was assessed using discrimination, calibration, and reclassification. A total of 2,300 eligible cases were enrolled. Of them, 288 (12.5%) patients reached composite outcome and 214 (9.3%) patients reached ESRD during a median follow-up period of 30 months. Using the composite outcome for analysis, the Clinical, Limited, Full, and CKD models had relatively good performance with similar C statistics (0.81, 0.81, 0.82, and 0.82, respectively). While using ESRD as the end point, the four prediction models had better performance (all C statistics > 0.9). Furthermore, subgroup analysis showed that the models containing clinical and pathological variables (Full model and Limited model) had better discriminatory abilities than the models including only clinical indicators (Clinical model and CKD model) in low-risk patients characterized by higher baseline eGFR (≥60 ml/min/1.73 m 2 ). In conclusion, we validated recently reported IgAN and CKD risk models in our Chinese IgAN cohort. Compared to pure clinical models, adding pathological variables will increase performance in predicting ESRD in low-risk IgAN patients with baseline eGFR ≥60 ml/min/1.73 m 2 ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ouyang, Zhao, Li, Luo, Xu, Shao, Chen, Yu, Jin, Xu, Shi, Hussain, Du, Fang, Pan, Wang, Xie and Chen.)

Background: Approximately 70% of congenital deafness is attributable to genetic causes. Incidence of congenital deafness is known to be higher in families with consanguineous marriage. In this study, we investigated the genetic causes in three consanguineous Pakistani families segregating with prelingual, severe-to-profound deafness., Results: Through targeted next-generation sequencing of 414 genes known to be associated with deafness, homozygous variants c.536del (p. Leu180Serfs∗20) in TECTA , c.3719 G>A (p. Arg1240Gln) in MYO7A , and c.482+1986&#95;1988del in HGF were identified as the pathogenic causes of enrolled families. Interestingly, in one large consanguineous family, an additional c.706G>A (p. Glu236Lys) variant in the X-linked POU3F4 gene was also identified in multiple affected family members causing deafness. Genotype-phenotype cosegregation was confirmed in all participating family members by Sanger sequencing., Conclusions: Our results showed that the genetic causes of deafness are highly heterogeneous. Even within a single family, the affected members with apparently indistinguishable clinical phenotypes may have different pathogenic variants., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Xueshuang Mei et al.)

Background: Joubert syndrome (JBTS) is a heterogenous disorder characterized by intellectual disability, developmental delays, molar tooth sign in brain imaging, hypotonia, ocular motor apraxia and overlapping features of ciliopathies. There are 36 clinical subtypes of JBTS, with an equal number of genes known so far for this phenotype., Methods: Whole exome sequencing (WES) and Sanger sequencing were performed for the molecular diagnosis of a Pakhtun family affected with Joubert syndrome type 9 (JBTS9)., Results: A novel homozygous missense variant (c.4417C>G; Pro1473Ala) in exon 34 was identified in coiled-coil and C2 domains-containing the protein 2A (CC2D2A; NM&#95;001080522) gene. The variant co-segregated in autosomal recessive fashion within the family and was not found in 200 ethnically matched unaffected individuals. In silico analyses supported the pathogenic effect of the altered CC2D2A protein., Conclusions: To the best of our knowledge, this is the first report of CC2D2A alteration co-segragating with a JBTS9 phenotype in a Pakhtun family from Pakistan. Our findings broaden the pathogenic spectrum of JBTS9, adding a novel variant to CC2D2A variation pool. WES analysis is a successful molecular diagnostic tool for rare genetic disorders, especially in those populations where the marriage of cousins is more frequent. Efficient and accurate genetic testing and counselling of the affected families are helpful for patient management and for reducing the disease burden in future generations., (© 2020 John Wiley & Sons, Ltd.)

Meiosis is pivotal for sexual reproduction and fertility. Meiotic programmed DNA double-strand breaks (DSBs) initiate homologous recombination, ensuring faithful chromosome segregation and generation of gametes. However, few studies have focused on meiotic DSB formation in human reproduction. Here, we report four infertile siblings born to a consanguineous marriage, with three brothers suffering from non-obstructive azoospermia and one sister suffering from unexplained infertility with normal menstrual cycles and normal ovary sizes with follicular activity. An autosomal recessive mutation in TOP6BL was found co-segregating with infertility in this family. Investigation of one male patient revealed failure in programmed meiotic DSB formation and meiotic arrest prior to pachytene stage of prophase I. Mouse models carrying similar mutations to that in patients recapitulated the spermatogenic abnormalities of the patient. Pathogenicity of the mutation in the female patient was supported by observations in mice that meiotic programmed DSBs failed to form in mutant oocytes and oocyte maturation failure due to absence of meiotic recombination. Our study thus illustrates the phenotypical characteristics and the genotype-phenotype correlations of meiotic DSB formation failure in humans., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest., (Copyright © 2020 Science China Press. Published by Elsevier B.V. All rights reserved.)

Background: Loss of function mutations in the spermine synthase gene (SMS) have been reported to cause a rare X-linked intellectual disability known as Snyder-Robinson Syndrome (SRS). Besides intellectual disability, SRS is also characterized by reduced bone density, osteoporosis and facial dysmorphism. SRS phenotypes evolve with age from childhood to adulthood., Methods: Whole exome sequencing was performed to know the causative gene/pathogenic variant. Later we confirmed the pathogenic variant through Sanger sequencing. Furthermore, we also performed the mutational analysis through HOPE SERVER and SWISS-MODEL. Also, radiographs were also obtained for affected individual to confirm the disease features., Results: In this article, we report the first Pakistani family consisting of three patients with SRS and a novel missense pathogenic variant in the SMS gene (c.905 C > T p.(Ser302Leu)). In addition to the typical phenotypes, one patient presented with early-onset seizures. Clinical features, genetic and in-silico analysis linked the affected patients of the family with Snyder-Robinson and suggest that this novel mutation affects the spermine synthase activity., Conclusion: A novel missense variant in the SMS, c.905C > T p. (Ser302Leu), causing Snyder- Robinson Syndrome (SRS) is reported in three members of Pakistani Family.

Background: Lipoprotein glomerulopathy (LPG) is a rare kidney disease caused by APOE mutations. The aim of this study was to correlate the genetic and clinical features of LPG., Methods: Totally eight LPG patients were recruited in this study and Sanger sequencing of APOE was performed for all available family members. Clinical and histological features were analyzed. A literature review of LPG was also conducted., Results: Genetic analysis revealed five patients with APOE-Kyoto, two with APOE-Osaka/Kurashiki, and one with APOE-Chicago mutations. LPG patients with urine protein reduced more than 50% had a slower decrease in renal function than those with less urine protein reduction (estimated glomerular filtration rate reduction rate -5.0 ± 0.8 vs. 1.5 ± 0.7 ml/min per 1.73 m 2 ⋅month -1 , p = .03). We then enrolled 95 LPG patients from previous studies and this study. LPG patients had higher blood pressure (mean arterial pressure: 109.4 ± 19.4 vs. 94.4 ± 11.1 mmHg, p < .001) than the control group. Interestingly, patients with APOE mutations in the LDL receptor binding region had higher serum apolipoprotein E (apoE) levels [ln(apoE): 2.7 ± 0.4 vs. 2.0 ± 0.5 mg/dl, p < .001] in comparison to other domains., Conclusion: Here, we report for the first time APOE-Osaka/Kurashiki and APOE-Chicago mutations in the Chinese population. LPG was associated with higher blood pressure and serum apoE levels were higher in patients with mutations in LDL receptor binding region. In addition, the findings further indicated that treatment of proteinuria might slow down renal function progression in these patients., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Lead ions (Pb 2+ ), one form of the toxic heavy metal, have drawn significant attention due to their harmful effects on human health and the environment. Although many analytical techniques have been developed over the past few decades, the development of a sensitive, selective, and rapid method to detect Pb 2+ remains a challenge. In this work, we developed a sensitive surface-enhanced Raman scattering (SERS) biosensor for highly sensitive detection of Pb 2+ by using DNAzyme-modified Fe 3 O 4 @Au@Ag nanoparticles (Fe 3 O 4 @Au@Ag NPs). Firstly, the thiolated 5'-Cy3 DNA probe was modified on the surface of Fe 3 O 4 @Au@Ag NPs, which hybridized with the Pb 2+ -specific DNAzyme to form a SERS biosensor, and the Cy3 labels were used to detect Pb 2+ . In the presence of Pb 2+ , the DNAzyme cleaves the Cy3-labeled DNA probe, leading to the release of Cy3-labeled DNA probe from the Fe 3 O 4 @Au@Ag NPs. Therefore, the Raman intensity of the Cy3 labels decreases. The proposed biosensor exhibited excellent linearity in the range from 0.01 to 1.0 nM, with a limit of detection for Pb 2+ of 5 pM. It features superior selectivity to Pb 2+ over other interfering metal ions and good application in the determination of Pb 2+ in tap water and human serum samples. The SERS biosensor provides a novel' simple and sensitive method for detection of Pb 2+ and sheds new light on the design and synthesis of analogous SERS biosensors for the detection of other heavy metal ions.

A novel surface-enhanced Raman scattering (SERS) analysis strategy has been designed combining Au@DTNB@Ag core-shell nanoparticles (DTNB attachment on gold nanoparticles, then encapsulated in Ag shell nanoparticles named as ADANPs) and duplex-specific nuclease signal amplification (DSNSA) platform. Firstly, ADANPs and magnetic substrate of Fe 3 O 4 nanoparticles were covalently attached to the 3'- and 5'- end of capture probe (CP) targeting miRNA-21. Upon the addition of target miRNA-21, these heteroduplexes were specifically cleaved by DSN and resulted in ADANPs that were released from the surface of Fe 3 O 4 nanoparticles (Fe 3 O 4 NPs). At the same time, miRNA-21 remained intact and can rehybridize another DNA probe to trigger the signal-amplifying reaction. Based on this principle, the developed SERS method exhibited good linearity in the range 0 to 1 nM for miRNA-21 with a limit of detection (LOD) of 0.084 fM and has an ability to differentiate even a single-base mismatched sequence on the target sequence or other miRNA sequence. The results provide a novel SERS method which can successfully been applied to the miRNA-21 detection in human serum. Graphical abstract a shows the synthesis of Fe3O4 NPs and the conjugation of Au@DTNB@Ag NPs (ADANPs) for the detection of miRNA-21, b shows the operating principle of DSN-assisted signal amplification strategy for miRNA detection based on Fe3O4@CP@ADA NPs.

Asthenozoospermia is a common cause of male infertility, but its etiology remains incompletely understood. We recruited three Pakistani infertile brothers, born to first-cousin parents, displaying idiopathic asthenozoospermia but no ciliary-related symptoms. Whole-exome sequencing identified a missense variant (c.G5408A, p.C1803Y) in DNAH17, a functionally uncharacterized gene, recessively cosegregating with asthenozoospermia in the family. DNAH17, specifically expressed in testes, was localized to sperm flagella, and the mutation did not alter its localization. However, spermatozoa of all three patients showed higher frequencies of microtubule doublet(s) 4-7 missing at principal piece and end piece than in controls. Mice carrying a homozygous mutation (Dnah17M/M) equivalent to that in patients recapitulated the defects in patients' sperm tails. Further examinations revealed that the doublets 4-7 were destabilized largely due to the storage of sperm in epididymis. Altogether, we first report that a homozygous DNAH17 missense variant specifically induces doublets 4-7 destabilization and consequently causes asthenozoospermia, providing a novel marker for genetic counseling and diagnosis of male infertility., (© 2019 Zhang et al.)

More than 2300 genes have been reported to be involved in spermatogenesis but the functional roles of most genes in male fertility remain to be elucidated. In this study, we explored the function of dipeptidase 3 (Dpep3), a gene predicted to be testis-specific, in male fertility of mice. We showed that Dpep3 is evolutionarily conserved in human and mouse along with other eutherians. Its mRNA was exclusively detected in testicular tissue and expressed in testes from 7 days postpartum. To further explore its role in male fertility, we generated Dpep3 knockout mice (Dpep3 -/- ) using the CRISPR/Cas9 technology and found that the male Dpep3 -/- mice are fertile despite a significant reduction in sperm count. Histology of testis and progression of meiotic prophase I showed no obvious difference between wild-type and Dpep3 -/- mice. All these findings indicate that Dpep3 is not essential for male fertility in mice. These findings will help other researchers to avoid research duplication, save their time and resources to focus on the genes that are indispensable for male fertility., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The role of cyclin-dependent kinase inhibitor 2A gene (CDKN2A) variants in breast cancer is not well understood, here we investigated their possible effects on breast cancer in Pakistani women attending the NORI Hospital, Islamabad. Direct DNA sequencing of CDKN2A identified an already known polymorphism in the 3' UTR, c.*29G>C (rs11515), in 5.88% patients and two novel variants. One, a deep intronic substitution (c.458-554T>G) in 1.96% patients, is also detected as a compound heterozygous form along with c.*29G>C in 1.96% patients (c.[458-554T>G; *29G>C]). The other is a novel deletion (c.458-82delG) occurring as a compound variant with two other identified variants c.[458-554T>G; 458-82delG; *29G>C] in 1.96% patients. In silico pathogenicity prediction analyses did not predict pathogenic effects on breast cancer for these individual variants. We conclude that variations in CDKN2A are not the major genetic cause of breast cancer in the enrolled Pakistani patients.

The balanced actions between ubiquitination and deubiquitination precisely control the levels of various proteins vital for spermatogenesis. Ubiquitin-specific processing proteases (USPs) are the largest family of deubiquitinatingenzymes(DUBs), containing more than 50 members. So far, the functions of only a few USPs in male fertility have been studied, the roles of the majority are yet unknown. The present study aimed to explore the function of Usp29 (ubiquitin-specific protease 29) in male fertility. We found that Usp29 showed predominant expression in mouse testis, and its mRNA expression started to increase at 14 days postpartum (dpp), with a peak at 28 and 35 dpp. Using CRISPR/Cas9 technology, we generated Usp29 knockout mice (Usp29 -/- ). Usp29 -/- mice exhibited no overt developmental anomalies. Further examination revealed that Usp29 -/- mice had normal fertility and showed no detectable difference in the testis/body weight ratio, testicular and epididymal histology as well as epididymal sperm count from the wild-type littermates. Moreover, Usp29 is not a pseudogene in mice. Taken together, our study first reported that though Usp29 is predominantly expressed in the testis, it is not essential for male fertility in mice.

Bromodomains are epigenetic readers of acetyl-lysine involved in chromatin remodeling and transcriptional regulations. Over the past few years, extensive research has been carried out to discover small-molecule inhibitors against bromodomains to treat various diseases. Cyclic AMP response element-binding protein (CREBBP) bromodomain has emerged as a hot target for cancer therapy. This study aims at discovering new inhibitors against CREBBP bromodomain using ligand-based molecular docking. A library of 2168 lead-like compounds were docked into the Kac binding site of CREBBP bromodomain. On the basis of the energy score and interaction analysis, six compounds were selected. In order to validate the stability of these six protein-ligand complexes 20 ns molecular dynamics simulations and principal component analyses were carried out. Based on the different analyses these six compounds may provide valuable information for developing CREBBP selective inhibitors.

In situ imaging of mRNA in living cells can help to monitor the real time mRNA expression and also useful for diagnosis and prognosis of the diseases. In this study, a new strategy was designed for simple, sensitive, and selective platform to detect the mRNA levels by combining a hairpin probe-graphene oxide (HP1/GO) and duplex-specific nuclease signal amplification (DSNSA). Initially, the DNA probe was adsorbed on the surface of GO to protect it from enzymatic digestion. Then, the target mRNA (T1) was hybridized with a partial hairpin probe which formed a duplex. Finally, under the action of DSN nuclease, the ssDNA in the DNA/RNA hybrid was selectively cleaved and produced small fragments. Then, T1 triggered the next reaction cycle, constituting a new circular exponential amplification. Here, we conclude that this assay is highly sensitive for the detection of target mRNA with the lower detection limit of 1 fM under optimal conditions. Furthermore, this strategy was successfully used for imaging of mRNA in living cells., (Copyright © 2018 Elsevier B.V. All rights reserved.)

CDKN2A was first identified as melanoma predisposition tumour suppressor gene and has been successively studied. The previous researches have not established any noteworthy association with breast cancer. Therefore, through extensive literature search and in-silico analysis, we have tried to focus on the role of CDKN2A in breast cancer. CDKN2A variants in breast cancer were collected from different databases. The overall percentage of variants (approximately 5.8%) and their incidence frequency in breast cancer cases were found to be very low as compared to the number of samples screened in different studies. Exon 2 was identified as the major region of alternations. Approximately 42.8% were entire gene deletions, while 24.2% were missense mutations. These variants cannot be ignored because of their pathogenic effects as interpreted by the bioinformatics tools used in the present study. Earlier studies have shown that CDKN2A excludes the predisposition of germline variants, but interestingly shares common breast cancer germline variants with other carcinomas. Most of the data have revealed this gene as rarely mutated or deleted in breast cancer. However, few association studies have shown that in addition to being a 'multiple' tumour suppressor gene, it is mutated/deleted more in breast cancer cell lines as compared to breast cancer tissues or blood samples; thus, this gene cannot be neglected as a breast cancer candidate gene. The deletion/malfunctioning of CDKN2A in different tumours including breast cancer has recently led to the discovery of many clinical CDK inhibitors. Furthermore, these collected genetic variants will also be helpful in developing diagnostic, preventive, and treatment approaches for patients.

Background: Congenital hypogonadotropic hypogonadism (CHH) is a heterogeneous disorder characterized by delayed or loss of puberty and infertility due to functional deficiency in the hypothalamic gonadotropin-releasing hormone (GnRH). CHH can be classified into 2 subtypes on the basis of olfaction: Kallmann syndrome and normosmic CHH (nCHH). The spectrum of genetic variants causing CHH is continually expanding. Here, we recruited a consanguineous Pakistani family having 2 male and 2 female infertile patients diagnosed with idiopathic nCHH., Aims: The aim of this study was to investigate the genetic cause of nCHH in the family., Methods: Clinical and physical analyses were performed for the patients. Genetic analysis was carried out using whole exome and Sanger sequencing., Results: Clinical and physical investigations confirmed low levels of gonadotropins and failure of secondary sexual development in the patients. Genetic analysis identified a novel nonsense mutation (chr4: g.68619942G>A, c.112C>T, p.Arg38*) in the gonadotropin-releasing hormone receptor gene (GNRHR) recessively co-segregating with nCHH in this family. All the patients are homozygous and their parents are heterozygous carriers, while normal siblings are heterozygous carriers or wild-type for this mutation, indicating that the identified mutation is pathogenic for nCHH in the family., Conclusion: We report the first homozygous nonsense mutation in the GNRHR gene (chr4: g. 68619942G>A, c.112C>T, p. Arg38*) that is associated with familial nCHH. Hence, our study displayed a good correlation of the genotype and phenotype of nCHH patients., (© 2019 S. Karger AG, Basel.)

The TP53 gene product, p53, is a pleiotropic transcription factor induced by stress, which functions to promote cell cycle arrest, apoptosis and senescence. Genome-wide profiling has revealed an extensive system of long noncoding RNAs (lncRNAs) that is integral to the p53 signalling network. As a research tool, we implemented a public access database called TP53LNC-DB that annotates currently available information relating lncRNAs to p53 signalling in humans.

Hereditary leukonychia (also known as porcelain nails or white nails) is a genetic disorder. It may exist as an isolated feature or associated with other cutaneous or systemic disorders. Although a number of genes have been described to cause leukonychia, still the underlying genetic etiologies of many cases remain unknown. Here, we report a Pakistani family presenting leukonychia and koilonychia nails in mother and five of her kids. All the affected individuals had white to pale nails in appearance exhibiting complete and partial leukonychia, respectively. Similarly, nails of finger and toe appeared brittle and concave, showing the characteristics features of koilonychia. Whole exome sequencing and subsequent Sanger sequencing identified a pathogenic novel missense mutation (c.1390G>A, p.Glu464Lys) in PLCD1, co-segregating with the disorder in an autosomal dominant pattern. In silico prediction tools supported the pathogenicity of the identified mutation. Literature review determined that mutations in PLCD1 only cause leukonychia. Therefore, our findings add another pathogenic variant to the PLCD1 mutation pool causing leukonychia that would help to understand the underlying molecular mechanism.

Background: Neurological disorders comprise a large group of clinically and genetically heterogeneous disorders, many of which have a genetic cause. In addition to a detailed neurological examination, exome sequencing is being increasingly used as a complementary diagnostic tool to identify the underlying genetic cause in patients with unclear, supposedly genetically determined disorders., Objective: To identify the genetic cause of a complex movement disorder in five consanguineous Pakistani families., Methods: We included five consanguineous Pakistani families with complex recessively inherited movement disorders. Clinical investigation including videotaping was carried out in a total of 59 family members (4-21 per family) and MRI in six patients. Exome sequencing was performed in 4-5 family members per pedigree to explore the underlying genetic cause., Results: Patients presented a wide spectrum of neurological symptoms including ataxia and/or dystonia. We identified three novel homozygous, segregating variants in ATCAY (p.Pro200Profs*20), MCOLN1 (p.Ile184Thr), and SACS (p.Asn3040Lysfs*4) in three of the families. Thus, we were able to identify the likely cause of the disease in a considerable number of families (60%) with the relatively simple and nowadays widely available method of exome sequencing. Of note, close collaboration of neurologists and geneticists was instrumental for proper data interpretation., Conclusions: We expand the phenotypic, genotypic, and ethnical spectrum of mutations in these genes. Our findings alert neurologists that rare genetic causes should be considered in complex phenotypes regardless of ethnicity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

There are more than 2300 genes that are predominantly expressed in mouse testes. The role of hundreds of these genes has been studied in mouse spermatogenesis but still there are many genes whose function is unknown. Gene knockout (KO) strategy in mice is widely used for in vivo study of gene function. The present study was designed to explore the function of the four genes: Tex37, Ccdc73, Prss55 and Nxt2, which were evolutionarily conserved in eutherians. We found that these genes had a testis-enriched expression pattern in mice except Nxt2. We knocked out these genes by CRISPR/Cas9 individually and found that all the KO mice had normal fertility with no detectable difference in testis/body weight ratios, epididymal sperm counts, as well as testicular and epididymal histology from wild type mice. Although these genes are evolutionarily conserved in eutherians including human and mouse, they are not individually essential for spermatogenesis, testis development and male fertility in mice in laboratory conditions. Our report of these fertile KO data could avoid the repetition and duplication of efforts which will help in prioritizing efforts to focus on genes that are indispensable for male reproduction.