Utility of labial salivary gland biopsy in the histological diagnosis of neuronal intranuclear inclusion disease.

BACKGROUND AND PURPOSE: Neuronal intranuclear inclusion disease (NIID) poses a diagnostic challenge because of its diverse clinical manifestations. Detection of intranuclear inclusions remains the primary diagnostic criterion for NIID. Skin biopsies have traditionally been used, but concerns exist regarding postoperative complications and scarring. We sought to investigate the diagnostic utility of labial salivary gland biopsy, a less invasive alternative. METHODS: This study included a total of 19 patients and 11 asymptomatic carriers who underwent labial gland biopsies, while 10 patients opted for skin biopsies. All these individuals were confirmed to have pathogenic GGC repeat expansions in the NOTCH2NLC gene. The control group comprised 20 individuals matched for age and sex, all with nonpathogenic GGC repeat expansions, and their labial gland tissue was sourced from oral surgery specimens. RESULTS: Labial gland biopsies proved to be a highly effective diagnostic method in detecting eosinophilic intranuclear inclusions in NIID patients. The inclusions showed positive staining for p62 and ubiquitin, confirming their pathological significance. The presence of uN2CpolyG protein in the labial gland tissue further supported the diagnosis. Importantly, all patients who underwent lip gland biopsy experienced fast wound healing without any noticeable scarring. In contrast, skin biopsies led to varying degrees of scarring and one instance of a localized infection. CONCLUSION: Labial salivary gland biopsy emerged as a minimally invasive, efficient diagnostic method for NIID, with rapid healing and excellent sensitivity.

Identification of the synonymous variant c.3141G > A in TNRC6B gene that altered RNA splicing by minigene assay.

BACKGROUND: Global developmental delay with speech and behavioral abnormalities (OMIM: 619243) is an autosomal dominant disease caused by variants in TNRC6B gene. METHOD: We reviewed and summarized clinical manifestations and genotypes in patients previously reported with TNRC6B gene variants. We used several prediction tools to predict pathogenicity and performed minigene assays to verify the function of the synonymous variant affecting RNA splicing. RESULT: The patient presented with convulsive seizures and developmental delay. WES combined with functional studies diagnosed a child with a synonymous variant in TNRC6B gene. Through minigene assay and Sanger sequencing, we demonstrated that c.3141G > A variant induced exon 7 skipping and the synonymous variant was pathogenic. CONCLUSION: Synonymous variants do not change the amino acids encoded by the codon, so we usually consider synonymous variants to be benign and ignore their pathogenicity. Minigene assay is a valuable tool to identify the effect of variation on RNA splicing and identify synonymous variants' benign or pathogenic. We showed that the synonymous variant was pathogenic by minigene assay. WES combined with minigene assay establishes a robust basis for genetic counseling and diagnosing diseases.

Mutation landscape of TSC1/TSC2 in Chinese patients with tuberous sclerosis complex.

Genetic testing of TSC1 and TSC2 is important for the diagnosis of tuberous sclerosis complex (TSC), an autosomal dominant neurocutaneous disease. This study retrospectively reviewed 347 samples from patients with clinically suspected TSC being tested for mutations in TSC1 and TSC2 genes using next-generation sequencing and multiplex ligation-dependent probe amplification. Two hundred eighty-one patients (80.98%) were classified as definite/possible/uncertain diagnosis of TSC and the mutational spectrum of TSC1/TSC2 was described. Two hundred eighteen unique nonsynonymous SNVs/Indels (64 in TSC1, 154 in TSC2) and 13 copy number variants (CNVs) were identified in 241 samples (85.77%), including 82 novel variants. CNVs involving 12 large deletions and one duplication were detected exclusively in TSC2. Both TSC1 and TSC2 mutations were nearly uniformly distributed in their protein-coding regions. Furthermore, a string of non-TSC1/TSC2 deleterious variants in 12 genes was identified in the patients, especially overwhelmingly present in the patients with no mutation identified (NMI) in TSC1/TSC2. Our study provides a comprehensive TSC1/TSC2 mutation landscape and reveal some potential risk non-TSCs variants present in patients with NMI.

Hepatic Autophagy Deficiency Compromises Farnesoid X Receptor Functionality and Causes Cholestatic Injury.

Autophagy is important for hepatic homeostasis, nutrient regeneration, and organelle quality control. We investigated the mechanisms by which liver injury occurred in the absence of autophagy function. We found that mice deficient in autophagy because of the lack of autophagy-related gene 7 or autophagy-related gene 5, key autophagy-related genes, manifested intracellular cholestasis with increased levels of serum bile acids, a higher ratio of tauromuricholic acid/taurocholic acid in the bile, increased hepatic bile acid load, abnormal bile canaliculi, and altered expression of hepatic transporters. In determining the underlying mechanism, we found that autophagy sustained and promoted the basal and up-regulated expression of farnesoid X receptor (Fxr) in the fed and starved conditions, respectively. Consequently, expression of Fxr and its downstream genes, particularly bile salt export pump, and the binding of FXR to the promoter regions of these genes, were suppressed in autophagy-deficient livers. In addition, codeletion of nuclear factor erythroid 2-related factor 2 (Nrf2) in autophagy deficiency status reversed the FXR suppression. Furthermore, the cholestatic injury of autophagy-deficient livers was reversed by enhancement of FXR activity or expression, or by Nrf2 deletion. Conclusion: Together with earlier reports that FXR can suppress autophagy, our findings indicate that autophagy and FXR form a regulatory loop and deficiency of autophagy causes abnormal FXR functionality, leading to the development of intracellular cholestasis and liver injury.

Mutation Analysis of Three Infantile Cases of X-linked Severe Combined Immunodeficiency.

BACKGROUND: Mutations in the IL2RG gene are known to cause X linked severe combined immunodeficiency (XSCID). More than 250 unique variants of the IL2RG gene have been reported to be identified in SCID patients so far, while many of them are of unknown significance which complicate the interpretation of mutation analysis results; furthermore, there are still many novel variants seen in clinical practice. METHODS: In this study we reviewed the testing results in three unrelated SCID families. All three probands had very severe immunodeficiency phenotypes and died in infancy. Next generation sequencing methods based on either SCID gene panel or exome sequencing were applied in causal variant screening for three probands. Sanger sequencing was used for verification of the variants of interest and carrier status study for the family members. STR analysis using the GoldeneyeTM DNA ID system (PeopleSpot Inc., China) was applied to verify the kinship of the family members when a de novo mutation was identified. RESULTS: Causal mutations were identified in all three SCID male probands, among which one was novel (c.557dupT), one was reported to be identified in a common variable immunodeficiency patient in literature (Leu87Pro), and one was a "hot-spot-mutation" (Arg226Cys). The patient with the missense mutation Leu87Pro in this study had much more severe infection phenotypes compared with the reported case in literature. CONCLUSIONS: Combining our findings and the published evidence together, Leu87Pro can be classified as a pathogenic variant following the ACMG guidelines. Correct and undoubted classification of the variants is of great importance for clinical gene testing.

Whole exome sequencing approach for identification of the molecular etiology in pediatric patients with hematuria.

BACKGROUND: Hematuria is a common condition in clinical practice of pediatric patients. It is related to a wide spectrum of disorders and has high heterogeneity both clinically and genetically, which contributes to challenges of diagnosis and lead many pediatric patients with hematuria not to receive accurate diagnosis and early management. METHODS: In this single center study, 42 children with hematuria were included in Tianjin Children's Hospital between 2019 and 2020. We analyzed the clinical information and performed WES (Whole exome sequencing) for all cases. Then the classification of identified variants was performed according to the American College of Medical Genetics and Genomics (ACMG) guidelines for interpreting sequence variants. For the fragment deletion, qPCR was performed to validate and confirm the inherited pattern. RESULTS: For the 42 patients, 16 cases had gross hematuria and 26 had microscopic hematuria. Molecular genetic causes were uncovered in 9 (21.4%) children, including 7 with Alport syndrome (AS), one with polycystic nephropathy and one with lipoprotein glomerulopathy. The genetic causes for other patients were not related with hematuria. CONCLUSIONS: WES is a rapid and effective way to evaluate patients with hematuria. The analysis of genotype-phenotype correlations of patients with AS indicated that severe variants were associated with early kidney failure. Secondary findings were not rare in Chinese children, thus the clinician should pay more attention to the clinical interpretation of sequencing results and properly interaction with patients and their family.

Identification of novel variants in hereditary spherocytosis patients by whole-exome sequencing.

Defects in erythrocyte membrane proteins can cause the most common type of inherited hemolytic anemia, so called hereditary spherocytosis (HS). It is characterized by the appearance of spherocytes in peripheral blood, hemolytic anemia, splenomegaly, jaundice and gallstones. Due to difficulty of diagnosis solely based on aforementioned parameters, the addition of genetic testing seems to be effective and most acknowledged. Up to date, pathogenic variations in five genes encoding membrane proteins (ANK1, SPTA1, SPTB, SLC4A1, EPB42) are identified to cause HS. Here, we have studied the genetic spectrum in forty-one patients with clinically suspected HS and their families, as well as their genotype-phenotype correlations. Pathogenic mutations in ANK1, SPTB, SLC4A1 and SPTA1 were found in 17 (41.5 %), 12 (29.3 %), 7 (17.1 %) and 5 (12.2 %) patients, respectively. Deleterious variants include 12 missense, 15 nonsense, 12 frameshift, and 4 splicing variants. Among these variations 32 were novel. In our genotype-phenotype analysis, platelet levels in SPTB (p = 0.021) and SLC4A1 (p = 0.02) patients were found to be significantly lower than ANK1 patients. In addition, LDH levels in SPTB patients were remarkably lower than patients with ANK1 mutations (p = 0.025).

A novel deletion of -2.8 kb removing the entire alpha 2-globin gene observed in a Chinese patient with Hb H.

Most of recognized alpha-thalassemia mutations include deletions of one or both alpha-globin genes. Here we describe a newly detected alpha-thalassemia-2 deletion characterized by a small 2.8 kb deletion involving the a 2 globin gene. This deletion has thus far been observed in one Chinese subject with hemoglobin H disease. Its breakpoints were detected to lie between coordinates 32485 and 35381 of the alpha-globin gene cluster (NG_000006.1), with a total of 2,894 nucleotides deleted. It was designated as -alpha2.8 deletion. The proband is a compound heterozygote deletion of -SEA and -alpha2.8, and the patient displayed very mild hemoglobin H disease phenotype with hemoglobin 9.8 g/dL.

Identification of two aberrant transcripts by RNA sequencing for a novel variant c.3354 + 5 G > A of MED12 in a Chinese girl with non-syndromic intellectual disability.

BACKGROUND: Missense variants in MED12 are associated with MED12-related disorders. We aimed to clarify the molecular level changes and underlying pathogenic mechanism of a female patient in our study. METHODS: We reported a Chinese girl with clinical characteristics similar to MED12-related disorders. Trio whole exome sequencing (WES) was performed to identify related pathogenic variant(s) and RNA sequencing (RNA-seq) was subsequently applied to evaluate the effect of identified variant(s) on mRNA splicing. Moreover, X-chromosome inactivation (XCI) assay based on AR and RP2 was performed to reveal the XCI pattern of the female patient. RESULTS: The proband manifested mainly as mental retardation and language impairment. Trio WES revealed a novel heterozygous variant c.3354 + 5 G > A in intron 23 of MED12. RNA-seq identified two aberrant transcripts. XCI assay on AR revealed a homozygous result, while XCI based on RP2 showed random pattern in peripheral blood. CONCLUSION: In conclusion, we identified a novel variant c.3354 + 5 G > A by WES combined with RNA-seq, which extends the spectrum of MED12 variants and provide a basis for further genetic counseling. According to the result of two aberrant transcripts by RNA-seq, we speculate that our patient's milder clinical feature may be the consequence of multiple different transcripts.

Identification of a Novel Deep Intronic Variant by Whole Genome Sequencing Combined With RNA Sequencing in a Chinese Patient With Menkes Disease.

Background: Menkes disease (MD) is a rare X-linked connective tissue disorder of copper metabolism caused by pathogenic variant(s) in ATP7A gene. The aim of the present study is to determine the clinical characteristics and molecular basis of one patient with MD. Methods: One 10-month-old Chinese boy who met the clinical manifestations of MD was enrolled in this study. Whole genome sequencing (WGS) was performed in the patient in order to identify the variant(s), followed by Sanger sequencing. RNA sequencing (RNA-seq) from whole blood was subsequently applied to assess the effect of variant on transcription levels, and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed for further validation. In addition, X chromosome inactivation (XCI) status of the patient's mother at the DNA level was measured by capillary electrophoresis. Results: The patient suffered from intermittent convulsions for more than 6 months, with psychomoto retardation and neurodegenerations. The patient also had curly hair, hypopigmented skin, cutis laxa, decreased muscle strength and hypotonia. MRI showed the intracranial arteries were tortuous with some "spiral" changes. The patient's serum ceruloplasmin level was low. WGS revealed one novel hemizygous variant, c.2627-501C > T (NM_000,052.7), located in the deep intronic sequence of ATP7A gene. Sanger sequencing confirmed that the variant was inherited from his mother. RNA-seq confirmed the variant itself, and identified a pseudo-exon inserted between exons 12 and 13 in mRNA of ATP7A. The sequencing results of RT-PCR from the patient confirmed this finding, while neither of his parents detected aberrant splicing. The Capillary electrophoresis results showed that the patient's mother had a skewed XCI. Conclusion: Our finding of the variant enlarges the variant spectrum in the ATP7A gene. This is a novel deep intronic variant which leads to the activation of a pseudo-exons in the ATP7A gene, and it demonstrates the usefulness of WGS combined with RNA-seq, in terms of revealing disease-causing variants in non-coding regions. Furthermore, the fact that the deep intronic variants cause disease by the activation of pseudo-exon inclusion indicates that in MD this might be an important mechanism.

Detection of α-globin gene deletion and duplication using quantitative multiplex PCR of short fluorescent fragments.

BACKGROUND: The aim of this study was to establish a sensitive method that can detect the presence of not only the common but also the unusual or unknown α-globin gene deletions for screening of α-thalassemia. We used quantitative multiplex PCR of short fluorescent fragments (QMPSF) for the α-globin genes (HBA) to screen α-thalassemia deletions. METHODS: We set up and validated HBA-QMPSF using 50 negative and 100 positive controls of deletional α-thalassemia. To evaluate its ability to detect the presence of the common and unusual or unknown α-globin gene deletions, 579 unrelated samples were simultaneously analyzed using this assay and multiplex Gap polymerase chain reaction (Gap-PCR). The inconsistent results were further confirmed by multiplex ligation-dependent probe amplification (MLPA). RESULTS: HBA-QMPSF was capable of detecting α-globin gene deletions with an acceptable variability as shown by mean values (SD) of allele dosage for the heterozygous deleted control obtained from intra- and inter-experimental replicates [0.63 (0.01) and 0.61 (0.03)]. In 572 out of the 579 unrelated subjects, HBA-QMPSF and multiplex Gap-PCR gave consistent results. In seven cases which were finally proved to be composed of one rare deletion--Thai/-α3.7, one novel deletion--SEA/-α2.8, four αααanti3.7/αα and one αααanti4.2/αα triplications, HBA-QMPSF showed deletion or duplication in the α-globin gene while multiplex Gap-PCR failed to give the correct diagnosis. CONCLUSIONS: HBA-QMPSF is able to detect the presence of the common and unusual or unknown α-thalassemia deletions and duplications. It can be used as an initial screening test for α-thalassemia caused by HBA gene copy number alteration.

Two cases of Fanconi-Bickel syndrome: first report from China with novel mutations of SLC2A2 gene.

Fanconi-Bickel syndrome (FBS) is a rare inherited disease caused by mutations in the glucose transporter 2 gene, SLC2A2. We reported the first two Chinese cases of FBS. Both cases presented typical clinical features of hepatomegaly, hypophosphatemic rickets, severely stunted growth, fasting hypoglycemia along with postprandial hyperglycemia, and proximal renal tubular dysfunction with disproportionately severe glucosuria. Genetic analysis of SLC2A2 gene revealed novel compound heterozygous mutations in both patients. The characteristics of being born as small for gestational age and apparent liver dysfunction in our cases have been seldom discussed in the literature. It seems FBS patients in general have lower birth weight than normal, but further data collection is still needed. Symptomatic treatments were effective, but the serum transaminase of patient 2 remained moderately increased, and he patient needed further follow-up. The present study will supplement the up-to-date clinical characteristic spectrum for FBS.

Hepatitis C virus genotypes and subtypes circulating in Mainland China.

The hepatitis C virus (HCV) exhibits global genotypic diversity. HCV genotyping plays an important role in epidemiological studies and clinical management. Herein, we report the results of HCV genotype and subtype detection in a large number of clinical samples, as performed by an independent laboratory in China. In total, four HCV genotypes and 18 subtypes were identified among 32 030 patients from 29 provinces and municipalities in China. Five dominant subtypes were detected from 98.84% of the samples: 1b (n=16 713, 52.18%), 2a (n=9188, 28.69%), 3b (n=2261, 7.06%), 6a (n=2052, 6.41%) and 3a (n=1479, 4.62%). Twelve rare subtypes were detected, of which four (that is, 6b, 6j, 6q and 6r) are reported for the first time in the Chinese population. Genotypes 4, 5 and 7 were not detected. Mixed infections of the dominant subtypes were found in a small portion of samples (n=65, 0.203%), in the following combinations: 1b-2a, 1b-3b, 1b-6a, 3a-3b, 1b-3a and 2a-6a. No mixed infections with rare subtypes were found. Males, compared with females, showed higher HCV subtype diversity, a lower percentage of HCV1b and 2a and a higher percentage of rare subtypes and mixed infections. Our analyses revealed the comprehensive distribution patterns of HCV genotypes in the general population of mainland China. HCV genotypic patterns were differentially distributed on the basis of geography, sex and age.

Gene Expression Analysis Indicates Divergent Mechanisms in DEN-Induced Carcinogenesis in Wild Type and Bid-Deficient Livers.

Bid is a Bcl-2 family protein. In addition to its pro-apoptosis function, Bid can also promote cell proliferation, maintain S phase checkpoint, and facilitate inflammasome activation. Bid plays important roles in tissue injury and regeneration, hematopoietic homeostasis, and tumorigenesis. Bid participates in hepatic carcinogenesis but the mechanism is not fully understood. Deletion of Bid resulted in diminished tumor burden and delayed tumor progression in a liver cancer model. In order to better understand the Bid-regulated events during hepatic carcinogenesis we performed gene expression analysis in wild type and bid-deficient mice treated with a hepatic carcinogen, diethylnitrosamine. We found that deletion of Bid caused significantly fewer alterations in gene expression in terms of the number of genes affected and the number of pathways affected. In addition, the expression profiles were remarkably different. In the wild type mice, there was a significant increase in the expression of growth regulation-related and immune/inflammation response-related genes, and a significant decrease in the expression of metabolism-related genes, both of which were diminished in bid-deficient livers. These data suggest that Bid could promote hepatic carcinogenesis via growth control and inflammation-mediated events.

Development of a novel DNA sequencing method not only for hepatitis B virus genotyping but also for drug resistant mutation detection.

BACKGROUND: In HBV-infected patients, different genotypes of the hepatitis B virus influence liver disease progression and response to antiviral therapy. Moreover, long-term antiviral therapy will eventually select for drug-resistant mutants. Detection of mutations associated to antiviral therapy and HBV genotyping are essential for monitoring treatment of chronic hepatitis B patients. RESULTS: In this study, a simple method of partial-S gene sequencing using a common PCR amplification was established for genotyping clinical HBV isolates sensitively, which could detect the drug-resistant mutations successfully at the same time. CONCLUSIONS: The partial S gene sequencing assay developed in this study has potential for application in HBV genotyping and drug resistant mutation detection. It is simpler and more convenient than traditional S gene sequencing, but has nearly the same sensitivity and specificity when compared to S gene sequencing.

[Polymorphism of DXS15, CA13, CA22 loci in Guangdong normal population].

The aim of this study was to investigate the polymorphism of microsatellite repeats DXS15, CA13, CA22 tightly linked to FVIII gene in Guangdong population and its practical value in genetic diagnosis for hemophilia A. The polymerase chain reaction (PCR) and capillary electrophoresis (CE) methods were adopted to test the variability of the 3 microsatellite repeat in Guangdong females, including 111 females, 222 X chromosomes for detecting DXS15 polymorphism; 87 females, 174X chromosomes for detecting CA13 polymorphism; 94 females, 188 X chromosomes for detecting CA22 polymorphism. The results indicated that 11 alleles corresponding to DXS15 were found at this locus with size ranging from 140 to 160 bp. The polymorphism information content (PIC) of this microsatellite repeat was 0.82, heterozygosity was 82%. Six alleles corresponding to CA13 were found, with a size from 145 to 155 bp, and PIC was 0.56, heterozygosity was 56.2%. Four alleles corresponding to CA22 were found with size ranging from 79 to 85 bp, and PIC was 0.41, heterozygosity was 50%. It is concluded that in contrast to the information about Caucasian, the polymorphism of these 3 microsatellites differs from race to race, and region to region. DXS15, CA13 and CA22 are highly polymorphic genetic markers useful for linkage analysis of haemophilia A, which may play a vital role in detection and prenatal diagnosis for hemophilia A.

[Screening of proteins binding to FXR1P using yeast two-hybrid technique].

OBJECTIVE: To screen the proteins interacting with FXR1P for functional investigation of FXR1P. METHODS: The yeast strain AH109 transformed with the recombinant expression vector pGBKT7/FXR1 was mated with the yeast strain Y187 pretransformed with human fetal brain cDNA library. The positive clones were screened and identified by sequence analysis. RESULTS: The recombinant expression vector pGBKT7/FXR1 was constructed successfully. Five proteins binding to FXR1P were screened from human fetal brain cDNA library using the yeast two-hybrid system, including CMAS, FTH1, GOLGA4, HSD17B1 and CSH1. CONCLUSIONS: These results provide new clues for investigating the biological functions of FXR1P and the pathogenesis of Fragile X syndrome.