[Analysis of the results for genetic disease screening among 1 000 newborns from Huzhou].

OBJECTIVE: To analyze the types and distribution of pathogenic variants for neonatal genetic diseases in Huzhou, Zhejiang Province. METHODS: One thousand neonates (48 ~ 42 h after birth) born to Huzhou region were selected as the study subjects. Dry blood spot samples were collected from the newborns, and targeted capture high-throughput sequencing was carried out for pathogenic genes underlying 542 inherited diseases. Candidate variants were verified by Sanger sequencing. RESULTS: Among the 1 000 newborns, the male to female ratio was 1.02 : 1.00. No pathogenic variants were detected in 253 cases, whilst 747 cases were found to carry at least one pathogenic variant, which yielded a carrier rate of 74.7%. The most frequently involved pathogenic gene was FLG, followed by GJB2, UGT1A1, USH2A and DUOX2. The variants were classified as homozygous, compound heterozygous, and hemizygous variants. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), 213 neonates were verified to have carried pathogenic and/or likely pathogenic variants, with a positive rate of 21.3%. The most commonly involved genes had included UGT1A1, FLG, GJB2, MEFV and G6PD. CONCLUSION: Newborn screening based on high-throughput sequencing technology can expand the scope of screening and improve the positive predictive value. Genetic counseling based on the results can improve the patients' medical care and reduce neonatal mortality and childhood morbidity, while provide assistance to family members' health management and reproductive decisions.

A Novel Mutation c.3392G>T of COL2A1 Causes Spondyloepiphyseal Dysplasia Congenital by Affecting Pre-mRNA Splicing.

Spondyloepiphyseal dysplasia congenital (SEDC) is a rare chondrodysplasia caused by dominant pathogenic variants in COL2A1. Here, we detected a novel variant c.3392G > T (NM_001844.4) of COL2A1 in a Chinese family with SEDC by targeted next-generation sequencing. To confirm the pathogenicity of the variant, we generated an appropriate minigene construct based on HeLa and HEK293T cell lines. Splicing assay indicated that the mutated minigene led to aberrant splicing of COL2A1 pre-mRNA and produced an alternatively spliced transcript with a skipping of partial exon 48, which generated a predicted in-frame deletion of 15 amino acids (p. Gly1131_Pro1145del) in the COL2A1 protein. Due to the pathogenicity of the variation, we performed prenatal diagnosis on the proband's wife, which indicated that the fetus carried the same mutation.

[Analysis of pathogenic variants of USH2A gene in a child with Usher syndrome type II].

OBJECTIVE: To detect pathogenic variant in a child featuring Usher syndrome type II. METHODS: Peripheral blood samples of the child and his parents were collected for the analysis of variants of hearing impairment-related genes. The findings were verified in 100 individuals with normal hearing. RESULTS: The child was found to harbor compound heterozygous variants of the USH2A gene, namely c.8224-1G>C in intron 41 and c.5678C>G(p.Ser1893X) in exon 28, which were inherited respectively from his mother and father. Based on the American College of Medical Genetics and Genomics standards and guidelines, both c.8224-1G>C and c.5678C>G(p.Ser1893X) variants of USH2A gene were predicted to be pathogenic(PVS1+PM2+PM3). CONCLUSION: The compound heterozygous variants c.8224-1G>C and c.5678C>G of the USH2A gene probably underlay the disease in this child. Above finding has enriched the spectrum of USH2A gene variants.

[Genetic analysis and prenatal diagnosis of a pregnant woman with Sheldon-Hall syndrome].

OBJECTIVE: To provide genetic testing and prenatal diagnosis for a woman with Sheldon-Hall syndrome. METHODS: The woman was subjected to targeted capture and next-generation sequencing for variant of genes associated with skeletal disorders. And the result was verified in her parents and fetus. RESULTS: The woman was found to harbor a c.188G>A variant of the TNNT3 gene, which was also found in her affected mother and the fetus. Her grandmother and grandmother's brother had similar manifestations, which was in line with an autosomal dominant inheritance. The same variant was not found in her father. CONCLUSION: The c.188G>A variant of the TNNT3 gene probably underlay the distal joint contracture in this pedigree, based on which prenatal diagnosis was attained.

Effects of immunomodulators on liquefaction and ulceration in the rabbit skin model of tuberculosis.

To better control tuberculosis (TB) epidemics in developing countries a real need exists to study the liquefaction and cavity formation that occur in pulmonary TB lesions. This report is the first to evaluate the effects of immunomodulators on these two processes in a rabbit skin model. The effects of recombinant human interferon-γ (rIFN-γ), recombinant human interleukin-2 (rIL-2), dexamethasone and cyclophosphamide (CTX) were evaluated in TB lesions produced by intradermal injection of 5 × 10(6) viable BCG bacilli. Recombinant IL-2 and rIFN-γ accelerated the liquefaction and healing of the lesions, and reduced the bacterial load. In contrast, dexamethasone inhibited the liquefaction of the lesions, and increased the bacterial load. The effect of CTX was similar to dexamethasone but not as pronounced. Serum levels of IL-2 were higher during the liquefaction and healing phases in the rIL-2 and rIFN-γ groups. Therefore, immunomodulators affect both the development of TB lesions and the survival of the mycobacteria within them. This study suggests that the rabbit skin model can be a valuable method to select therapeutic agents that could inhibit liquefaction and cavity formation in pulmonary tuberculosis.

Construction and evaluation of a multistage Mycobacterium tuberculosis subunit vaccine candidate Mtb10.4-HspX.

To search for more effective vaccines to enhance the immunogenicity and protective efficacy of Mycobacterium bovis Bacille Calmette-Guerin (BCG) and to control or even eradicate Mycobacterium tuberculosis (M. tuberculosis) in all stages of infection including the persister bacteria, antigens of Mtb10.4 (Rv0288) expressed in replicating bacilli and HspX (also called Acr, Hsp16.3, Rv2031c) highly expressed in dormant bacilli were fused together to construct a multistage fusion protein Mtb10.4-HspX (MH for short) without affinity tag with potential advantage for clinical use. The human T-cell responses to MH were evaluated for its immunogenicity. Furthermore, MH was emulsified in an adjuvant composed of N,N'-dimethyl-N,N'-dioctadecylammonium bromide (DDA) and mycobacterial cord factor trehalose-6,6-dimycolate (TDM) to construct subunit vaccine, whose immunogenicity and potency to boost BCG primed immunity against M. tuberculosis infection were evaluated in mice. The results showed that the fusion protein MH without affinity tag was stably produced in Escherichia coli and was successfully purified by chromatography. MH was strongly recognized by human T cells from TB patients and persons latently infected with M. tuberculosis. In conclusion, MH in adjuvant DDA-TDM generated strong antigen-specific humoral and cell-mediated immunity, and had the capability to enhance BCG-primed immunity and the protective efficacy against M. tuberculosis in mice. These findings suggest that MH in DDA-TDM have the potential to be a good multistage tuberculosis vaccine candidate.

Observation of the density and size of cells in hippocampus and vascular lesion in thalamus of GFAP-apoE transgenic mice.

OBJECTIVE: Apolipoprotein E (apoE) is associated with increased risk of age-related diseases, such as Alzheimer's disease (AD) and cerebrovascular disease (CVD). The present study aims to investigate the age-related general morphological changes of the brain in GFAP-apoE transgenic mice, especially the alterations in number and size of hippocampal pyramidal cells and the microvascular lesions in the thalamus. METHODS: Nine female apoE4/4 mice were divided into 3 groups (n=3 in each group): 3-4 months (young group), 9-10 months (middle-aged group) and 20-21 months (old group). Age-matched apoE3/3 mice were employed as control group (n=3 in each group). The paraffin sections of brain tissue were stained by 2 conventional staining methods, thionin staining and hematoxylin-esion(HE) staining, the former of which was to observe the hippocampal cells, while the latter was used to examine the brain microvasculature. RESULTS: There was no apparent difference in the cortical layer between apoE3/3 and apoE4/4 mice, neither any significant difference in the number of cells in hippocampal CA1-CA3 subfields between apoE3/3 and apoE4/4 mice at various age points (P>0.05). However, the mean size of pyramidal cells in CA1 subfield in apoE3/3 and apoE4/4 mice decreased as mice were getting older (P<0.001). At the age of 20-21 months, this cellular atrophy in apoE4/4 mice was more severe than that in old apoE3/3 mice (P<0.05). Furthermore, microvascular lesion in the thalamus was detected in all the 3 old apoE4/4 mice, at varying degrees (5.24%, 1.41% and 3.97%, respectively), while only one apoE3/3 mouse exhibited microvascular lesion in the thalamus, at a low level (0.85%). CONCLUSION: The current study suggests that the cell size in hippocampal CA1 subfield decreases with aging, irrespective of apoE genotype. Cellular atrophy in CA1 subfield and the microvascular lesion in the thalamus are both more severe in old apoE4/4 mice as compared with those in age-matched apoE3/3 mice. Doubts still exist on whether the decreased cell size in hippocampal CA1 subfield in old apoE4/4 mice is associated with dysfunction in learning and memory and whether the microvascular lesions indicate a higher risk of stroke in human apoE4 allele mice. To clarify these issues, further investigations are needed.