Genome sequencing demonstrates high diagnostic yield in children with undiagnosed global developmental delay/intellectual disability: A prospective study.

Genome sequencing (GS) has been used in the diagnosis of global developmental delay (GDD)/intellectual disability (ID). However, the performance of GS in patients with inconclusive results from chromosomal microarray analysis (CMA) and exome sequencing (ES) is unknown. We recruited 100 pediatric GDD/ID patients from multiple sites in China from February 2018 to August 2020 for GS. Patients have received at least one genomic diagnostic test before enrollment. Reanalysis of their CMA/ES data was performed. The yield of GS was calculated and explanations for missed diagnoses by CMA/ES were investigated. Clinical utility was assessed by interviewing the parents by phone. The overall diagnostic yield of GS was 21%. Seven cases could have been solved with reanalysis of ES data. Thirteen families were missed by previous CMA/ES due to improper methodology. Two remained unsolved after ES reanalysis due to complex variants missed by ES, and a CNV in untranslated regions. Follow-up of the diagnosed families revealed that nine families experienced changes in clinical management, including identification of targeted treatments, cessation of unnecessary treatment, and considerations for family planning. GS demonstrated high diagnostic yield and clinical utility in this undiagnosed GDD/ID cohort, detecting a wide range of variant types of different sizes in a single workflow.

HPDL deficiency causes a neuromuscular disease by impairing the mitochondrial respiration.

Mitochondrial diseases are caused by variants in both mitochondrial and nuclear genomes. A nuclear gene HPDL (4-hydroxyphenylpyruvate dioxygenase-like), which encodes an intermembrane mitochondrial protein, has been recently implicated in causing a neurodegenerative disease characterized by pediatric-onset spastic movement phenotypes. Here, we report six Chinese patients with bi-allelic HPDL pathogenic variants from four unrelated families showing neuropathic symptoms of variable severity, including developmental delay/intellectual disability, spasm, and hypertonia. Seven different pathogenic variants are identified, of which five are novel. Both fibroblasts and immortalized lymphocytes derived from patients show impaired mitochondrial respiratory function, which is also observed in HPDL-knockdown (KD) HeLa cells. In these HeLa cells, overexpression of a wild-type HPDL gene can rescue the respiratory phenotype of oxygen consumption rate. In addition, a decreased activity of the oxidative phosphorylation (OXPHOS) complex II is observed in patient-derived lymphocytes and HPDL-KD HeLa cells, further supporting an essential role of HPDL in the mitochondrial respiratory chain. Collectively, our data expand the clinical and mutational spectra of this mitochondrial neuropathy and further delineate the possible disease mechanism involving the impairment of the OXPHOS complex II activity due to the bi-allelic inactivations of HPDL.

Loss of SPACA1 function causes autosomal recessive globozoospermia by damaging the acrosome-acroplaxome complex.

STUDY QUESTION: Is the sperm acrosome membrane-associated protein 1 (SPACA1) gene critical to human globozoospermia? SUMMARY ANSWER: The biallelic loss-of-function (variant of SPACA1) causes globozoospermia as a result of acrosome-acroplaxome complex damage. WHAT IS KNOWN ALREADY: SPACA1 expression decreases in patients with globozoospermia. Spaca1 gene-disrupted mice have abnormally shaped sperm heads that resemble those of human globozoospermia. STUDY DESIGN, SIZE, DURATION: We recruited a consanguineous family with two brothers affected by infertility as a consequence of globozoospermia. The semen analysis data and ART outcomes were collected. Exome sequencing (ES) was used to identify potential pathogenic variants. Protein-protein interaction (PPI) technologies and proteomic analysis were utilized to explore the pathogenic mechanism. PARTICIPANTS/MATERIALS, SETTING, METHODS: Two globozoospermic brothers and their consanguineous parents were recruited to identify the potential pathogenic variant through ES. A homozygous nonsense variant in the SPACA1 gene in both brothers inherited from the heterozygous parents was identified. Twenty normal fertile males were recruited as controls. Sperm ultrastructure was observed with transmission electron microscopy. Western blotting was performed to measure SPACA1 expression level in the sperm from the patients. Mass spectrometry (MS) analyses were used to identify differentially expressed proteins and to investigate proteins that interact with SPACA1. Co-immunoprecipitation (co-IP), yeast two-hybrid (Y2H) and immunofluorescence colocalization assays were used to confirm the PPI. MAIN RESULTS AND THE ROLE OF CHANCE: A nonsense variant (NM_030960.2: c.53G>A; p. Trp18*) in the SPACA1 gene was identified as the pathogenic variant in a family with globozoospermia. Patient IV:1 and Patient IV:2 had a phenotype very similar to that of Spaca1 gene-disrupted mice. The nonsense variant in SPACA1 led to premature transcriptional termination in the signal peptide, which was confirmed by western blotting. MS-based proteomics analysis showed that eight interactors of SPACA1 were differentially expressed in the patients' sperm, including actin-like Protein 7A (ACTL7A), an important component of the acrosome-acroplaxome complex. The PPI of SPACA1 and ACTL7A was confirmed via co-IP and Y2H assays. Immunofluorescence showed that SPACA1 and ACTL7A colocalized in mature sperm, revealing that these proteins were coexpressed spatially. LIMITATIONS, REASONS FOR CAUTION: Given the rarity of globozoospermia, only two patients from one family harbouring the SPACA1 variant were found. Future studies should evaluate SPACA1 variants in larger cohorts to corroborate this finding. WIDER IMPLICATIONS OF THE FINDINGS: This study revealed that the SPACA1 gene was critical for globozoospermia, which expanded the spectrum of causative genes for globozoospermia. This study also provided evidence for ICSI clinical outcomes for patients with SPACA1-deficient globozoospermia, which may guide clinical treatment strategies. Furthermore, this study explored the pathogenesis of globozoospermia caused by SPACA1 deficiency. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the Precision Medical Research of National Key Research and Development Program (2018YFC1002400), National Natural Science Foundation of China (81873724), and Natural Science Foundation of Shanghai (20ZR1472700). The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Dual diagnosis of osteogenesis imperfecta (OI) and short stature and advanced bone age with or without early-onset osteoarthritis and/or osteochondritis dissecans (SSOAOD) reveals a cumulative effect on stature caused by mutations in COL1A1 and ACAN genes.

Short stature is a feature when a person's height is more than 2 SDS below the corresponding mean height for a given age, gender and population. It can be influenced by many factors essential to growth plate. Here we report a three-generation family with 13 patients affected by osteogenesis imperfecta (OI) type I, short stature and advanced bone age, with or without early-onset osteoarthritis and/or osteochondritis dissecans (SSOAOD) or both. Panel sequencing of the proband revealed mutations in two extracellular matrix related genes: COL1A1 and ACAN. When comparing the quantitative trait, height within the family of different mutation carrier groups, we found an interesting cumulative effect, the ones with both mutations manifest shortest stature. Dual diagnoses of the family also suggest the necessity of a comprehensive molecular diagnosis method, such as panel/exome sequencing, especially encountering patients with novel phenotype or extreme trait.

RT-PCR analysis of mRNA revealed the splice-altering effect of rare intronic variants in monogenic disorders.

BACKGROUND: Variants perturbing the normal splicing of pre-mRNA can lead to human diseases. The splice-altering effect and eventual consequence on gene function was sometimes uncertain and hinders a definitive molecular diagnosis. METHODS: The impact of four rare intronic variants on splicing was analyzed through reverse transcription - polymerase chain reaction (RT-PCR) analysis of mRNA derived from the peripheral blood of patients. The results were compared with in-silico prediction. Potential implication on molecular diagnosis was discussed. RESULTS: Four rare intronic variants of SLC9A6, DLG3, GAA, and OCRL were identified in patients with suspected disorders, respectively. Although these four variants were all predicted to alter splicing by in-silico tools, RT-PCR analysis of mRNA derived from peripheral blood showed these variants affected splicing in different ways: c.899+3_899+6del of SLC9A6 resulted in one-exon skipping and an out-of-frame transcript; c.905-2A > G of DLG3 resulted in a mix of in-frame transcripts; c.1195-11T > A of GAA resulted in the in-frame insertion of nine nucleotides; c.723-2A > C of OCRL resulted in one-exon skipping and in-frame deletion of 102 nucleotides. The consequence revealed by mRNA analysis is essential for accurate interpretation of pathogenicity. CONCLUSION: Four intronic variants all caused aberrant mRNA splicing. For intronic variants with uncertain impact on splicing, mRNA analysis is helpful for ascertainment of alternative splicing and accurate interpretation of pathogenicity.

Whole Genome Low-Coverage Sequencing Concurrently Detecting Copy Number Variations and Their Underlying Complex Chromosomal Rearrangements by Systematic Breakpoint Mapping in Intellectual Deficiency/Developmental Delay Patients.

Simple copy number variations (CNVs) detected by chromosomal microarray (CMA) can result from complex structural changes. Therefore, it is necessary to characterize potential structural changes that cause pathogenic CNVs. We applied whole-genome low-coverage sequencing (WGLCS) to concurrently detect pathogenic CNVs and their associated chromosomal rearrangements in 15 patients. All the patients had an average of 2-3 pathogenic CNVs involving 1-2 chromosomes. WGLCS identified all the 34 pathogenic CNVs found by microarray. By identifying chimeric read pairs, WGLCS mapped 70 breakpoints in these patients, of which 47 were finely mapped at the nucleotide level and confirmed by subsequent PCR amplification and Sanger sequencing of the junction fragments. In 15 patients, structural rearrangements were defined at molecular level in 13 patients. In 13 patients, WGLCS reveal no additional results in two patients. In another 11 patients, WGLCS revealed new breakpoints or finely mapped the genes disrupted by breakpoints or 1-6 bp microhomology and/or short insertion (4-70 bp) in the breakpoints junctions. However, structural changes in the other two patients still remained unclear after WGLCS was performed. The structural alteration identified in the 13 patients could be divided into the following categories: (1) interstitial inverted duplication with concomitant terminal deletion (inv dup del) (P1,P4,P9,P11); (2) the product of pericentric inversion (P5); (3) ring chromosome (P8); (4) interstitial duplication and/or triplication (P6, P7); and (5) +der(22)t(11;22) (P2,P15); (6) complex structural rearrangements (P3,P12,P14). WGLCS displayed the ability to discover CNVs and define breakpoints and its disrupted genes and its surrounding sequences in one experiment at base-pair-resolution, which help us to learn more about the mechanisms of formation of observed genomic rearrangements, and in which DNA replicative/repair mechanism might contribute to the formation of complex rearrangements in 11 patients. Clear karyotype at molecular level could help provide an accurate evaluation of recurrent risk and guide prenatal diagnosis or reproductive planning.

High Detection Rate of Copy Number Variations Using Capture Sequencing Data: A Retrospective Study.

BACKGROUND: Capture sequencing (CS) is widely applied to detect small genetic variations such as single nucleotide variants or indels. Algorithms based on depth comparison are becoming available for detecting copy number variation (CNV) from CS data. However, a systematic evaluation with a large sample size has not been conducted to evaluate the efficacy of CS-based CNV detection in clinical diagnosis. METHODS: We retrospectively studied 3010 samples referred to our diagnostic laboratory for CS testing. We used 68 chromosomal microarray analysis-positive samples (true set [TS]) and 1520 reference samples to build a robust CS-CNV pipeline. The pipeline was used to detect candidate clinically relevant CNVs in 1422 undiagnosed samples (undiagnosed set [UDS]). The candidate CNVs were confirmed by an alternative method. RESULTS: The CS-CNV pipeline detected 78 of 79 clinically relevant CNVs in TS samples, with analytical sensitivity of 98.7% and positive predictive value of 49.4%. Candidate clinically relevant CNVs were identified in 106 UDS samples. CNVs were confirmed in 96 patients (90.6%). The diagnostic yield was 6.8%. The molecular etiology includes aneuploid (n = 7), microdeletion/microduplication syndrome (n = 40), and Mendelian disorders (n = 49). CONCLUSIONS: These findings demonstrate the high yield of CS-based CNV. With further improvement of our CS-CNV pipeline, the method may have clinical utility for simultaneous evaluation of CNVs and small variations in samples referred for pre- or postnatal analysis.

A Novel Nonsense Mutation of PHF6 in a Female with Extended Phenotypes of Borjeson-Forssman-Lehmann Syndrome

Borjeson-Forssman-Lehmann syndrome (BFLS) is a rare X-linked disease caused by PHF6 mutations. Classic BFLS has been associated with intellectual disability (ID), developmental delay (DD), obesity, epilepsy, typical facial features and anomalies of fingers and toes. Endocrinological phenotypes and outcome of treatment in this condition remain to be delineated. Here we report a patient who exhibited complete growth hormone deficiency who responded to hormonal treatment but with adverse effects. Horseshoe kidney was present in this patient, which is also atypical in BFLS. A heterozygous nonsense mutation c.673C>T (p.R225X) of PHF6 gene was identified in the patient, inherited from her unaffected mother. Both the patient and her mother showed highly skewed X-inactivation. We reviewed the phenotypes of all reported BFLS cases, and summarized their endocrine presentations. This first report of an Asian patient with BFLS further delineated the genetic and phenotypic spectrum of the syndrome. The adverse effect experienced by the patient suggests caution in the use of growth hormone treatment in this condition.

Biallelic UNC80 mutations caused infantile hypotonia with psychomotor retardation and characteristic facies 2 in two Chinese patients with variable phenotypes.

Biallelic UNC80 mutations cause infantile hypotonia with psychomotor retardation and characteristic facies 2 (IHPRF2), which is characterized by hypotonia, developmental delay (DD)/intellectual disability (ID), intrauterine growth retardation, postnatal growth retardation and characteristic facial features. We report two unrelated Chinese patients with compound heterozygous UNC80 mutations inherited from their parents, as identified by whole-exome sequencing (WES). Mutations c.3719G>A (p.W1240*)/c.4926_4937del (p.N1643_L1646del) and c.4963C>T (p.R1655C)/c.8385C>G (p.Y2795*) were identified in patient 1 and patient 2, respectively. Although both patients presented with DD/ID and hypotonia, different manifestations also occurred. Patient 1 presented with infantile hypotonia, epilepsy and hyperactivity without growth retardation, whereas patient 2 presented with persistent hypotonia, growth retardation and self-injury without epilepsy. Furthermore, we herein summarize the genotypes and phenotypes of patients with UNC80 mutations reported in the literature, revealing that IHPRF2 is a phenotypically heterogeneous disease. Common facial dysmorphisms include a thin upper lip, a tented upper lip, a triangular face, strabismus and microcephaly. To some extent, the manifestations of IHPRF2 mimic those of Angelman syndrome (AS)-like syndromes.

Rottlerin exhibits anti-cancer effect through inactivation of S phase kinase-associated protein 2 in pancreatic cancer cells.

Rottlerin, a natural product isolated from Mallotus philippinensis, has been characterized as an effective chemoprevention agent in inhibiting tumor cell growth. Although multiple studies have revealed the role of rottlerin in tumorigenesis, the molecular mechanism of rottlerin-mediated anti-tumor activity has not been fully elucidated. It has been reported that Skp2 (S-phase kinase associated protein 2) plays an oncogenic role in human malignancies, indicating that inactivation of Skp2 could be a promising approach for the treatment of cancers. Therefore, in this study, we aim to investigate whether rottlerin exhibits its anti-tumor activities via targeting Skp2 pathway in pancreatic cancer. We found that rottlerin inhibited cell growth, induced apoptosis, arrested cell cycle, and retarded cell invasion and migration. Notably, we observed that the expression of Skp2 was significantly decreased in rottlerin-treated pancreatic cancer cells. Importantly, overexpression of Skp2 abrogated the anti-tumor function induced by rottlerin in pancreatic cancer cells. Consistently, depletion of Skp2 promoted rottlerin-mediated inhibition of cell growth and invasion. Collectively, our study demonstrated that rottlerin could suppress Skp2 expression and subsequently exert its tumor suppressive function in pancreatic cancer cells, suggesting that rottlerin might be a potential therapeutic compound for treating pancreatic cancer.

Rottlerin inhibits cell growth and invasion via down-regulation of Cdc20 in glioma cells.

Rottlerin, isolated from a medicinal plant Mallotus phillippinensis, has been demonstrated to inhibit cellular growth and induce cytoxicity in glioblastoma cell lines through inhibition of calmodulin-dependent protein kinase III. Emerging evidence suggests that rottlerin exerts its antitumor activity as a protein kinase C inhibitor. Although further studies revealed that rottlerin regulated multiple signaling pathways to suppress tumor cell growth, the exact molecular insight on rottlerin-mediated tumor inhibition is not fully elucidated. In the current study, we determine the function of rottlerin on glioma cell growth, apoptosis, cell cycle, migration and invasion. We found that rottlerin inhibited cell growth, migration, invasion, but induced apoptosis and cell cycle arrest. Mechanistically, the expression of Cdc20 oncoprotein was measured by the RT-PCR and Western blot analysis in glioma cells treated with rottlerin. We observed that rottlerin significantly inhibited the expression of Cdc20 in glioma cells, implying that Cdc20 could be a novel target of rottlerin. In line with this, over-expression of Cdc20 decreased rottlerin-induced cell growth inhibition and apoptosis, whereas down-regulation of Cdc20 by its shRNA promotes rottlerin-induced anti-tumor activity. Our findings indicted that rottlerin could exert its tumor suppressive function by inhibiting Cdc20 pathway which is constitutively active in glioma cells. Therefore, down-regulation of Cdc20 by rottlerin could be a promising therapeutic strategy for the treatment of glioma.

Rottlerin exerts its anti-tumor activity through inhibition of Skp2 in breast cancer cells.

Studies have investigated the tumor suppressive role of rottlerin in carcinogenesis. However, the molecular mechanisms of rottlerin-induced anti-tumor activity are largely unclear. Skp2 (S-phase kinase associated protein 2) has been validated to play an oncogenic role in a variety of human malignancies. Therefore, inactivation of Skp2 could be helpful for the treatment of human cancers. In the current study, we explore whether rottlerin could inhibit Skp2 expression, leading to inhibition of cell growth, migration and invasion in breast cancer cells. We found that rottlerin treatment inhibited cell growth, induced apoptosis and cell cycle arrest. We also revealed that rottlerin suppressed cell migration and invasion in breast cancer cells. Mechanically, we observed that rottlerin significantly down-regulated the expression of Skp2 in breast cancer cells. Importantly, overexpression of Skp2 abrogated rottlerin-mediated tumor suppressive activity, whereas down-regulation of Skp2 enhanced rottlerin-triggered anti-tumor function. Strikingly, we identified that rottlerin exhibited its anti-tumor potential partly through inactivation of Skp2 in breast cancer. Our findings indicate that rottlerin could be a potential safe agent for the treatment of breast cancer.

SCF(ß-TRCP) promotes cell growth by targeting PR-Set7/Set8 for degradation.

The Set8/PR-Set7/KMT5a methyltransferase plays critical roles in governing transcriptional regulation, cell cycle progression and tumorigenesis. Although CRL4(Cdt2) was reported to regulate Set8 stability, deleting the PIP motif only led to partial resistance to ultraviolet-induced degradation of Set8, indicating the existence of additional E3 ligase(s) controlling Set8 stability. Furthermore, it remains largely undefined how DNA damage-induced kinase cascades trigger the timely destruction of Set8 to govern tumorigenesis. Here, we report that SCF(ß-TRCP) earmarks Set8 for ubiquitination and degradation in a casein kinase I-dependent manner, which is activated by DNA-damaging agents. Biologically, both CRL4(Cdt2) and SCF(ß-TRCP)-mediated pathways contribute to ultraviolet-induced Set8 degradation to control cell cycle progression, governing the onset of DNA damage-induced checkpoints. Therefore, like many critical cell cycle regulators including p21 and Cdt1, we uncover a tight regulatory network to accurately control Set8 abundance. Our studies further suggest that aberrancies in this delicate degradation pathway might contribute to aberrant elevation of Set8 in human tumours.

Curcumin suppresses cell growth and invasion and induces apoptosis by down-regulation of Skp2 pathway in glioma cells.

Studies have demonstrated that curcumin exerts its tumor suppressor function in a variety of human cancers including glioma. However, the exact underlying molecular mechanisms remain obscure. Emerging evidence has revealed that Skp2 (S-phase kinase associated protein 2) plays an oncogenic role in tumorigenesis. Therefore, we aim to determine whether curcumin suppresses the Skp2 expression, leading to the inhibition of cell growth, invasion, induction of apoptosis, and cell cycle arrest. To this end, we conducted multiple methods such as MTT assay, Flow cytometry, Wound healing assay, invasion assay, RT-PCR, Western blotting, and transfection to explore the functions and molecular insights of curcumin in glioma cells. We found that curcumin significantly inhibited cell growth, suppressed cell migration and invasion, induced apoptosis and cell cycle arrest in glioma cells. Furthermore, we observed that overexpression of Skp2 promoted cell growth, migration, and invasion, whereas depletion of Skp2 suppressed cell growth, migration, and invasion and triggered apoptosis in glioma cells. Mechanistically, we defined that curcumin markedly down-regulated Skp2 expression and subsequently up-regulated p57 expression. Moreover, our results demonstrated that curcumin exerts its antitumor activity through inhibition of Skp2 pathway. Collectively, our findings suggest that targeting Skp2 by curcumin could be a promising therapeutic approach for glioma prevention and therapy.

Arsenic Trioxide Inhibits Cell Growth and Invasion via Down- Regulation of Skp2 in Pancreatic Cancer Cells.

Arsenic trioxide (ATO) has been found to exert anti-cancer activity in various human malignancies. However, the molecular mechanisms by which ATO inhibits tumorigenesis are not fully elucidated. In the current study, we explored the molecular basis of ATO-mediated tumor growth inhibition in pancreatic cancer cells. We used multiple approaches such as MTT assay, wound healing assay, Transwell invasion assay, annexin V-FITC, cell cycle analysis, RT-PCR and Western blotting to achieve our goal. We found that ATO treatment effectively caused cell growth inhibition, suppressed clonogenic potential and induced G2-M cell cycle arrest and apoptosis in pancreatic cancer cells. Moreover, we observed a significant down-regulation of Skp2 after treatment with ATO. Furthermore, we revealed that ATO regulated Skp2 downstream genes such as FOXO1 and p53. These findings demonstrate that inhibition of Skp2 could be a novel strategy for the treatment of pancreatic cancer by ATO.

NEDD4: a promising target for cancer therapy.

The Neuronally expressed developmentally downregulated 4 (NEDD4), functioning largely as an E3 ubiquitin ligase, has been demonstrated to play a critical role in the development and progression of human cancers. In this review, to understand the regulatory mechanism(s) of NEDD4 as well as the signaling pathways controlled by NEDD4, we briefly describe the NEDD4 upstream regulators and its downstream ubiquitin substrates. Moreover, we further discuss its oncogenic roles in human malignancies. Therefore, targeting NEDD4 could be a potential therapeutic strategy for treatment of human cancers.

Aberrant regulation of FBW7 in cancer.

FBW7 (F-box and WD repeat domain-containing 7) or Fbxw7 is a tumor suppressor, which promotes the ubiquitination and subsequent degradation of numerous oncoproteins including Mcl-1, Cyclin E, Notch, c- Jun, and c-Myc. In turn, FBW7 is regulated by multiple upstream factors including p53, C/EBP-δ, EBP2, Pin1, Hes-5 and Numb4 as well as by microRNAs such as miR-223, miR-27a, miR-25, and miR-129-5p. Given that the Fbw7 tumor suppressor is frequently inactivated or deleted in various human cancers, targeting FBW7 regulators is a promising anti-cancer therapeutic strategy.