Clinical analysis of Gabriele-de Vries caused by YY1 mutations and literature review.

BACKGROUND: Gabriele-de Vries syndrome is a rare autosomal dominant genetic disease characterized by global development delay/intellectual disability, delayed language development, feeding difficulties, and distinctive facial dysmorphism. It is caused by pathogenic variants in YY1. METHODS: The current report describes a female patient with motor delay and a facial dysmorphism phenotype. We identified pathogenic mutations in the patient by whole-exome sequencing and confirmed them by Sanger sequencing. RESULTS: A novel heterozygous frameshift mutation NM_003403.5:c.458_476del (p. V153fs*97) in the YY1 gene was detected in the proband. Finally, we provide a case-based review of the clinical features associated with Gabriele-de Vries syndrome. A total of 28 patients with genetic abnormalities and clinical phenotypes have been reported in the literature thus far. CONCLUSIONS: The mutation site is reported for the first time, and its discovery would expand the mutation spectrum of the YY1 gene. The main clinical manifestations of Gabriele-de Vries syndrome are developmental delay/intellectual disability, craniofacial dysplasia, intrauterine growth delay, low birth weight, feeding difficulties, and rare congenital malformations. Genetic tests are crucial techniques for its diagnosis because of its nonspecific clinical manifestations.

Atypical retinopathy in ataxia with vitamin E deficiency: report of a sibship.

Typical retinitis pigmentosa (RP) may not be the only retinal phenotype encountered in ataxia with vitamin E deficiency (AVED). The following short case series describes a novel form of retinopathy in AVED. We describe two patients with AVED belonging to the same consanguineous sibship. Both presented an unusual retinopathy consisting of scattered, multifocal, nummular, hyperautofluorescent atrophic retinal patches. The retinopathy remained stable under vitamin E supplementation. We hypothesize these changes to be the result of arrested AVED-related RP following early supplementation with α-tocopherol acetate.

Unraveling the Role of Molecular Profiling in Predicting Treatment Response in Stage III Colorectal Cancer Patients: Insights from the IDEA International Study.

BACKGROUND: This study aimed to investigate the molecular profiles of 237 stage III CRC patients from the international IDEA study. It also sought to correlate these profiles with Toll-like and vitamin D receptor polymorphisms, clinicopathological and epidemiological characteristics, and patient outcomes. METHODS: Whole Exome Sequencing and PCR-RFLP on surgical specimens and blood samples, respectively, were performed to identify molecular profiling and the presence of Toll-like and vitamin D polymorphisms. Bioinformatic analysis revealed mutational status. RESULTS: Among the enrolled patients, 63.7% were male, 66.7% had left-sided tumors, and 55.7% received CAPOX as adjuvant chemotherapy. Whole exome sequencing identified 59 mutated genes in 11 different signaling pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) CRC panel. On average, patients had 8 mutated genes (range, 2-21 genes). Mutations in ARAF and MAPK10 emerged as independent prognostic factors for reduced DFS (p = 0.027 and p < 0.001, respectively), while RAC3 and RHOA genes emerged as independent prognostic factors for reduced OS (p = 0.029 and p = 0.006, respectively). Right-sided tumors were also identified as independent prognostic factors for reduced DFS (p = 0.019) and OS (p = 0.043). Additionally, patients with tumors in the transverse colon had mutations in genes related to apoptosis, PIK3-Akt, Wnt, and MAPK signaling pathways. CONCLUSIONS: Molecular characterization of tumor cells can enhance our understanding of the disease course. Mutations may serve as promising prognostic biomarkers, offering improved treatment options. Confirming these findings will require larger patient cohorts and international collaborations to establish correlations between molecular profiling, clinicopathological and epidemiological characteristics and clinical outcomes.

De Novo Mutation in KRT1 Leads to Epidermolytic Palmoplantar Keratoderma: from Chinese Traditional Treatment to Prenatal Diagnosis Using Whole-Exome Sequencing-Plus.

Congenital skin disorders are a class of complex genetic diseases that are difficult to diagnose and treat. We developed trio whole-exome sequencing-plus (WES-plus) for detecting de novo mutations and evaluated the use of traditional Chinese medicine (TCM) for treating congenital skin disorders. In this study, we successively performed panel-based next-generation sequencing (NGS) and Trio WES-plus in a child with frequent large blisters. Panel-based NGS revealed no pathogenic mutations. Trio WES-plus for resequencing based on cutaneous keratosis of the palms and feet detected a missense mutation (c.1436T>A, p.Ile479Asn) in the coding region of KRT1 in the child but not in his parents. Following prenatal diagnosis, a healthy second baby without the mutation was born. The disease symptoms of epidermolytic palmoplantar keratoderma (EPPK) application were improved by TCM and Western medicine. Our study revealed the pathogenicity of a de novo mutation in human KRT1, which expands the mutation spectrum of EPPK. Trio WES-plus is useful for diagnosing genetic diseases and providing genetic guidance from prenatal diagnosis to treatment.

Autoantibody Positivity in Two Bahraini Siblings With a Novel Alpha-Methylacyl-CoA Racemase Mutation.

Bile acid synthesis disorders (BASD) are a group of rare autosomal recessive disorders. Of the nine different versions, BASD type 4 is characterized by a gene mutation in alpha-methylacyl-CoA racemase (AMACR), which is located on chromosome 5p13. These disorders generally present with a normal gamma-glutamyl transferase with cholestasis, absence of pruritis, and malabsorption of fat, which can lead to fat-soluble vitamin deficiencies. In adulthood, patients usually develop neurological sequelae. Initial testing can be done through the measurement of urine metabolites; however, confirmation of the diagnosis is achieved through whole exome sequencing. Treatment involves supplementation of oral cholic acid and modification of diet. Only 23 patients with this disease have been described. Here, we report two cases of siblings from a family in Bahrain with a novel AMACR mutation and a unique association with autoimmune antibodies alongside a literature review.

De novo CLPTM1 variants with reduced GABAA R current response in patients with epilepsy.

OBJECTIVE: To investigate the clinical features and potential pathogenesis mechanism of de novo CLPTM1 variants associated with epilepsy. METHODS: Identify de novo genetic variants associated with epilepsy by reanalyzing trio-based whole-exome sequencing data. We analyzed the clinical characteristics of patients with these variants and performed functional in vitro studies in cells expressing mutant complementary DNA for these variants using whole-cell voltage-clamp current recordings and outside-out patch-clamp recordings from transiently transfected human embryonic kidney (HEK) cells. RESULTS: Two de novo missense variants related to epilepsy were identified in the CLPTM1 gene. Functional studies indicated that CLPTM1-p.R454H and CLPTM1-p.R568Q variants reduced the γ-aminobutyric acid A receptor (GABAA R) current response amplitude recorded under voltage clamp compared to the wild-type receptors. These variants also reduced the charge transfer and altered the time course of desensitization and deactivation following rapid removal of GABA. The surface expression of the GABAA R γ2 subunit from the CLPTM1-p.R568Q group was significantly reduced compared to CLPTM1-WT. SIGNIFICANCE: This is the first report of functionally relevant variants within the CLPTM1 gene. Patch-clamp recordings showed that these de novo CLPTM1 variants reduce GABAA R currents and charge transfer, which should promote excitation and hypersynchronous activity. This study may provide insights into the molecular mechanisms of the CLPTM1 variants underlying the patients' phenotypes, as well as for exploring potential therapeutic targets for epilepsy.

Feline precision medicine using whole-exome sequencing identifies a novel frameshift mutation for vitamin D-dependent rickets type 2.

OBJECTIVES: A 14-week-old female domestic longhair kitten presented with shifting lameness and disproportionately smaller size compared with a co-housed littermate. METHODS: Hematology and serum biochemical testing were conducted to investigate causes for delayed growth, and radiographs of the appendicular skeleton were obtained. RESULTS: The afflicted kitten had marked hypocalcemia, mild hypophosphatemia and substantial elevations in alkaline phosphatase activity, as well as pathognomonic radiographic findings consistent with rickets. Skeletal changes and hypocalcemia prompted testing of concentrations of parathyroid hormone (PTH) and vitamin D metabolites. Endocrine testing demonstrated significant increases in serum concentrations of PTH and 1,25-dihydroxycholecalciferol (calcitriol), supporting a diagnosis of vitamin D-dependent rickets type 2. Provision of analgesia, supraphysiologic doses of calcitriol and calcium carbonate supplementation achieved normalization of the serum calcium concentration and restoration of normal growth, although some skeletal abnormalities persisted. Once skeletally mature, ongoing calcitriol supplementation was not required. Whole-exome sequencing (WES) was conducted to identify the underlying DNA variant. A cytosine deletion at cat chromosome position B4:76777621 in VDR (ENSFCAT00000029466:c.106delC) was identified and predicted to cause a stop codon in exon 2 (p.Arg36Glufs*18), disrupting >90% of the receptor. The variant was unique and homozygous in this patient and absent in the sibling and approximately 400 other cats for which whole-genome and whole-exome data were available. CONCLUSIONS AND RELEVANCE: A unique, heritable form of rickets was diagnosed in a domestic longhair cat. WES identified a novel frameshift mutation affecting the gene coding for the vitamin D3 receptor, determining the likely causal genetic variant. Precision medicine techniques, including whole-exome and whole-genome sequencing, can be a standard of care in cats to identify disease etiologies, and to target therapeutics and personalize treatment.

First Reported Case of Gabriele-de Vries Syndrome with Spinal Dysraphism.

Gabriele-de Vries syndrome is a rare autosomal dominant genetic disease caused by de novo pathogenic variants in the Yin Yang 1 (YY1) gene. Individuals with this syndrome present with multiple congenital anomalies, as well as a delay in development and intellectual disability. Herein, we report the case of a newborn male patient with a novel de novo pathogenic variant in the Guanine Nucleotide-Binding Protein, Alpha Stimulating (GNAS) gene, which was identified by whole-exome sequencing. Our patient suffered from a large open spinal dysraphism which was treated surgically immediately after birth. During the follow-up, facial dysmorphism, bladder and bowel incontinence, and mildly delayed motor and speech development were observed. Congenital central nervous system disorders were also confirmed radiologically. In this case report, we present our diagnostic and treatment approaches to this patient. To our knowledge, this is the first reported case of Gabriele-de Vries syndrome presenting with spinal dysraphism. Extensive genetic evaluation is the cornerstone in treatment of patients with suspected Gabriele-de Vries syndrome. However, in cases with potentially life-threatening conditions, surgery should be strongly considered.

An Extraordinary EEG Phenomenon Misdiagnosed as Nonconvulsive Status Epilepticus: Frequent Subclinical Periodic Discharges Terminated by Sudden Auditory Stimuli.

Triggering or modulation of seizures and rhythmic EEG patterns by external stimuli are well-known with the most common clinical appearance of stimulus induced periodic discharges (SI- PDs) patterns which are elicited by physical or auditory stimulation. However, stimulus terminated periodic discharges (ST-PDs), in other words, the periodic discharges stopped by external stimuli is an extremely rare electroencephalographic (EEG) finding. We report a 20-year-old woman with a marked psychomotor developmental delay of unknown cause, with frequent EEG patterns of long-lasting (10-60 s) bilateral paroxysmal high-voltage slow waves with occasional spikes, misdiagnosed as non-convulsive status epilepticus. However, no apparent clinical change was noted by the technician, physician, and her mother during these subclinical ictal EEG recordings. Interestingly, however, these epileptic discharges were abruptly interrupted by sudden verbal stimuli on the EEG, repeatedly. Whole exome sequencing and genotyping were performed to investigate possible genetic etiology that revealed two sequence variants, a frameshift variant of CACNA1H NM_021098.3:c.1701del;p.Asp568ThrfsTer15 and a missense variant of GRIN2D NM_000836.4:c.1783A>T;p.Thr595Ser as well as a copy number variant part deletion of ATP6V1A gene arr [hg19]3q13.31(113,499,698_113,543,081)x1 as possible pathogenic candidates. The subclinical periodic discharges terminated by verbal stimuli, is a very rare manifestation and needs particular attention. External modulation of ictal-appearing EEG patterns is important to identify stimulus terminated EEG patterns.

Integrative analysis of multiomics data identifies selenium-related gene ALAD associating with keshan disease.

Keshan disease is an endemic fatal dilated cardiomyopathy that can cause heart enlargement, heart failure, and cardiogenic death. Selenium deficiency is considered to be the main cause of Keshan disease. However, the molecular mechanism underlying Keshan disease remains unclear. Our whole-exome sequencing from 68 patients with Keshan disease and 100 controls found 199 candidate genes by gene-level burden tests. Interestingly, using multiomics data, the selenium-related gene ALAD (δ-aminolevulinic acid dehydratase) was the only candidate causative gene identified by three different analysis approaches. Based on single-cell transcriptome data, ALAD was highly expressed in cardiomyocytes and double mutations of human ALAD dramatically reduced its enzyme activity in vitro compared to negative control. Functional analysis of ALAD inhibition in mice resulted in a Keshan phenotype with left ventricular enlargement and cardiac dysfunction, whereas administration of sodium selenite markedly reversed the changes caused by ALAD inhibition. In addition, sodium selenite reversed Keshan phenotypes by affecting energy metabolism and mitochondrial function in mice as shown by the transcriptomic and proteomic data and the ultrastructure of cardiac myocytes. Our findings are the first to demonstrate that the selenium-related gene ALAD is essential for cardiac function by maintaining normal mitochondrial activity, providing strong molecular evidence supporting the hypothesis of selenium deficiency in Keshan disease. These results identified ALAD as a novel target for therapeutic intervention in Keshan disease and Keshan disease-related dilated cardiomyopathy.

Synovitis, Acne, Pustulosis, Hyperostosis, and Osteitis (SAPHO) Syndrome with Cutis Verticis Gyrata: Case Report and Review of Literature.

SAPHO (synovitis, acne, pustulosis, hyperostosis, and osteitis) syndrome is a rare disease clinically characterized by a wide range of cutaneous and osteoarticular manifestations. Here, we report a case of SAPHO syndrome with cutis verticis gyrata (CVG) and investigated the genetic causes in the four members of this pedigree. After failure of conventional treatments, a recombinant human TNF-α receptor II:IgG Fc fusion protein (rhTNFR:Fc, YISAIPU®) achieved good control of the disease at the 2-year follow-up. We did not identify any pathogenic mutation in this pedigree. We also summarized the clinical and therapeutic characteristics of 83 patients with SAPHO syndrome through the China National Knowledge Infrastructure (CKNI) database from 2016 to 2021. Patients with acne were young and predominantly male. About 45.8% patients were treated with biological therapies or traditional Chinese medicine (TCM), 84.2% of which showed positive effects against cutaneous and osteoarticular manifestations. We report a case of SAPHO syndrome with CVG that was successfully treated with rhTNFR:Fc. Our results reveal the genetic heterogeneity involved. Biologics and TCM are likely alternative options for the treatment of SAPHO syndrome.

A de novo YY1 missense variant expanding the Gabriele-de Vries syndrome phenotype and affecting X-chromosome inactivation.

Yin and Yang 1 gene (YY1; MIM#600,013) is recognized as a dual transcriptional activating and repressing factor, RNA-binding protein, and 3D chromatin regulator, with multi roles in neurodevelopmental and maintenance pathways. YY1 haploinsufficiency caused either by heterozygous sequence variants or deletions involving the whole gene has been recently associated with Gabriele-de Vries syndrome (GADEVS), a rare congenital autosomal dominant condition, leading to intellectual disability (ID) and multiple physical/behavioural abnormalities. Herein, we describe clinical and molecular findings from a Brazilian female harbouring a de novo missense pathogenic variant in YY1 gene (NM_003403.5:c.1106A > G; p.Asn369Ser) found by whole exome sequencing with potential implications for protein structure and function. Undescribed or uncommon clinical features in this patient included non-febrile seizures, severe scoliosis, hearing impairment, and chorioretinitis. Further bioinformatics analyses using YY1-other protein interaction networks reinforced the involvement of YY1 interactors in such phenotypes, in exception of chorioretinitis. Moreover, X-chromosome inactivation (XCI) skewing was evidenced in the patient and attributed to the haploinsufficiency of YY1, which direct and indirectly interacts with numerous XCI key regulators. Besides expanding the mutational and phenotype spectrum of GADEVS, our results highlight the role of YY1 as an essential autosomal regulator of XCI epigenetic process.

A Unique Mechanism of a Novel Synonymous PHEX Variant Causing X-Linked Hypophosphatemia.

CONTEXT: Synonymous mutations are usually nonpathogenic. OBJECTIVE: We report here a family with X-linked hypophosphatemia (XLH) due to a novel synonymous PHEX variant with a unique mechanism. METHODS: We studied a 4-member family (a mother, a son, and 2 daughters), all affected with XLH. Genomic DNA was extracted from peripheral leucocytes. Whole exome sequencing (WES) was used to identify the underlying genetic variant in the proband (the son). Sanger sequencing was used to confirm this variant in the proband and his family members. RT-PCR and sequencing of the cDNA revealed the effect of this variant on the PHEX structure and function. RESULTS: A synonymous variant in the PHEX gene (c.1701A>C) was identified in all affected members. This variant changes the first nucleotide of exon 17 from adenine to cytosine. Using RT-PCR, this variant was shown to interfere with splicing of exons 16 with 17 resulting in a single shorter PHEX transcript in the proband compared to normal control. Sanger sequencing of the cDNA revealed a complete skipping of exon 17 and direct splicing of exons 16 and 18. This led to a frameshift and an introduction of a new stop codon in the next codon (codon 568), which ultimately led to truncation and loss of the final 183 amino acids of PHEX. CONCLUSION: This novel variant shows how a synonymous exonic mutation may induce a complex series of changes in the transcription and translation of the gene and causes a disease, a mechanism that is not commonly recognized.

Clinical features of patients with Yin Yang 1 deficiency causing Gabriele-de Vries syndrome: A new case and review of the literature.

BACKGROUND: Gabriele-de Vries syndrome is a rare autosomal dominant genetic disease caused by de novo pathogenic variants in YY1. In this study, we report a 10-year-old boy with a de novo novel pathogenic variant in YY1, the first Iranian patient with Gabriele-de Vries Syndrome. METHODS: The novel de novo pathogenic variant detected in this study (NM_003403:c.690delA, p.Glu231Ilefs*25) was identified by whole-exome sequencing and confirmed by Sanger sequencing. RESULTS: The proband presented with delayed motor and speech development, ataxia, abnormal gait, autistic behavior, brain atrophy, and severe learning disability. Finally, we provide a case-based review of the clinical features associated with Gabriele-de Vries Syndrome. Thus far, merely 13 Gabriele-de Vries Syndrome patients have been reported in the literature. CONCLUSION: The investigations for a suspected case of Gabriele-de Vries Syndrome must involve molecular diagnosis of the disease and its underlying genetic defect because the clinical investigations are generally variable and nonspecific.

Patterns of Whole Exome Sequencing in Resected Cholangiocarcinoma.

BACKGROUND: With minimally effective chemotherapy options, cholangiocarcinoma patients have 5 year survival rate of 10%. Tumor genetic profiling (TGP) can identify mutations susceptible to targeted therapies. We sought to describe the use of TGP and frequency of actionable results in resected cholangiocarcinoma. METHODS: A retrospective review of patients undergoing curative intent resection at a comprehensive cancer center (2010-2020). Clinicopathologic and partial or whole exome sequencing data were reviewed. RESULTS: 114 patients (mean age 65 ± 11 years, 45% female) underwent resection of cholangiocarcinoma (46% poorly differentiated, 54% intrahepatic, 36% node positive, 75% margin negative). Additionally, 32% of patients underwent TGP, yielding a mean of 3.1 actionable mutations per patient (range 0-14). Mutations aligned with a median of one drug per patient (range 0-11). Common mutations included TP53 (33%), KRAS (31%), IDH1/2 (14%), FGFR (14%), and BRAF (8%). Targeted therapies were administered in only 4% of patients (23% of eligible sequenced patients). After a median 22 months, 23% had recurrence and 29% were deceased. DISCUSSION: TGP for cholangiocarcinoma has increased over the last decade with targeted therapies identified in most sequenced tumors, impacting treatment in a quarter of eligible patients. Precision medicine will play a central role in the future care of cholangiocarcinoma.

Novel Treatment for Congenital Disorder of Glycosylation in a Patient with Novel Homozygote Mutation of PMM2: A Case Report and Review Literature.

BACKGROUND: In Congenital Disorder of Glycosylation (CDG) type Ia, homozygous mutations of the PMM2 gene cause phosphomannomutase 2 dysfunction. CASE PRESENTATION: Herein, a 10-month-old girl, is presented with severe hypotonia, along with inappropriately normal mental status and normal facies. High 2-ketoglutaric acid was detected in her urine, therefore, the diagnosis of 2-Ketoglutarate dehydrogenase complex (KDHC) deficiency was made for this patient. A high dose of vitamin B1 was administered because thiamine is considered a co-factor in this inborn error of metabolism. She responded very well to the daily administration of 500 mg/day vitamin B1 and stood up without help 5 months later. She had also experienced a seizure, which responded well to pyridoxine. Then, she grew up into a 3.5-years-old child who could talk and walk normally. Recently, whole-exome sequencing was performed for her, which showed homozygote mutation of PMM2, therefore, the diagnosis was changed from KDHC deficiency to PMM2-CDG. CONCLUSION: Paying attention to the pathophysiology of inborn errors of metabolism is necessary while considering the defective enzyme co-factor, which may help us to find an option for the treatment of such rare diseases.

Hints from a Female Patient with Breast Cancer Who Later Presented with Cowden Syndrome.

A 51-year-old woman presented with metachronous tumor development in bilateral breasts, thyroid, and endometrium. Additional signs and symptoms fulfilled the National Comprehensive Cancer Network criteria for Cowden syndrome. Immunohistochemistry showed loss of PTEN expression in all tumors. Single nucleotide variants, 647 germline variants (including one each in PTEN and MSH3), and 21 somatic mutations within exons were detected in all tumors after whole-exome sequencing. There were 0, 11, and 46 specific somatic mutations in bilateral breasts, thyroid, and endometrial cancers, respectively. Although PTEN mutation is key to the development of Cowden syndrome, DNA repair dysfunction might be the initial driver of mutations. Fewer mutations were required to induce initial bilateral breast carcinomas, with subsequent thyroid and endometrial carcinomas requiring more mutations for induction. When genetic screening is unavailable, breast cancer patients with clinical manifestations of Cowden syndrome must be carefully assessed for secondary malignancies, such as thyroid and endometrial carcinomas.

Genomic characteristics and drug screening among organoids derived from non-small cell lung cancer patients.

BACKGROUND: Patient-derived organoid (PDO) models are highly valuable and have potentially widespread clinical applications. However, limited information is available regarding organoid models of non-small cell lung cancer (NSCLC). This study aimed to characterize the consistency between primary tumors in NSCLC and PDOs and to explore the applications of PDOs as preclinical models to understand and predict treatment response during lung cancer. METHODS: Fresh tumor samples were harvested for organoid culture. Primary tumor samples and PDOs were analyzed via whole-exome sequencing. Paired samples were subjected to immunohistochemical analysis. There were 26 antineoplastic drugs tested in the PDOs. Cell viability was assessed using the Cell Titer Glo assay 7-10 days after drug treatment. A heatmap of log-transformed values of the half-maximal inhibitory concentrations was generated on the basis of drug responses of PDOs through nonlinear regression (curve fit). A total of 12 patients (stages I-III) were enrolled, and 7 paired surgical tumors and PDOs were analyzed. RESULTS: PDOs retained the histological and genetic characteristics of the primary tumors. The concordance between tumors and PDOs in mutations in the top 20 NSCLC-related genes was >80% in five patients. Sample purity was significantly and positively associated with variant allele frequency (Pearson r = 0.82, P = 0.0005) and chromosome stability. The in vitro response to drug screening with PDOs revealed high correlation with the mutation profiles in the primary tumors. CONCLUSIONS: PDOs are highly credible models for detecting NSCLC and for prospective prediction of the treatment response for personalized precision medicine. KEY POINTS: Lung cancer organoid models could save precious time of drug testing on patients, and accurately select anticancer drugs according to the drug sensitivity results, so as to provide a powerful supplement and verification for the gene sequencing.

A Synonymous Variant c.579A>G in the ETFDH Gene Caused Exon Skipping in a Patient With Late-Onset Multiple Acyl-CoA Dehydrogenase Deficiency: A Case Report.

Background: Multiple acyl-CoA dehydrogenase deficiency (MADD) is an autosomal recessive disorder characterized by a wide range of clinical features, including muscle weakness, hypoglycemia, metabolic acidosis, and multisystem dysfunctions. Loss-of-function mutations in the electron transfer flavoprotein dehydrogenase (ETFDH) gene are associated with MADD. Disease-causing synonymous variants in the ETFDH gene have not been reported so far. Methods: We reported the clinical course of a Chinese girl who was diagnosed with late-onset MADD by the whole exome sequencing. The effects of variants on mRNA splicing were analyzed through transcript analysis in vivo and minigene splice assay in vitro. Results: The 6-month-old girl initially showed muscle weakness, muscular hypotonia, mild myogenic damage, and fatty liver. The blood and urine metabolic screening by tandem mass spectrometry suggested MADD. Molecular analysis of ETFDH gene revealed two novel heterozygous variants, a frameshift mutation c.1812delG (p.V605Yfs*34) in exon 13 and a synonymous variant c.579A>G (p.E193E) in exon 5. The transcript analysis in vivo exhibited that the synonymous variant c.579A>G caused exon 5 skipping. The minigene splice assay in vitro confirmed the alteration of ETFDH mRNA splicing which could lead to the production of a truncated protein. Supplementation of riboflavin, carnitine and low-fat diet improved the clinical symptoms. Conclusion: We firstly report a rare case of MADD with a pathogenic synonymous variant in the ETFDH gene which highlights the importance and necessity of bioinformatic analysis and functional testing for synonymous variants when searching for causative gene mutations. The results expand the spectrum of pathogenic variants in MADD.

Applications and analysis of targeted genomic sequencing in cancer studies.

Next Generation Sequencing (NGS) has dramatically improved the flexibility and outcomes of cancer research and clinical trials, providing highly sensitive and accurate high-throughput platforms for large-scale genomic testing. In contrast to whole-genome (WGS) or whole-exome sequencing (WES), targeted genomic sequencing (TS) focuses on a panel of genes or targets known to have strong associations with pathogenesis of disease and/or clinical relevance, offering greater sequencing depth with reduced costs and data burden. This allows targeted sequencing to identify low frequency variants in targeted regions with high confidence, thus suitable for profiling low-quality and fragmented clinical DNA samples. As a result, TS has been widely used in clinical research and trials for patient stratification and the development of targeted therapeutics. However, its transition to routine clinical use has been slow. Many technical and analytical obstacles still remain and need to be discussed and addressed before large-scale and cross-centre implementation. Gold-standard and state-of-the-art procedures and pipelines are urgently needed to accelerate this transition. In this review we first present how TS is conducted in cancer research, including various target enrichment platforms, the construction of target panels, and selected research and clinical studies utilising TS to profile clinical samples. We then present a generalised analytical workflow for TS data discussing important parameters and filters in detail, aiming to provide the best practices of TS usage and analyses.