Targeting strategy and generation of Sidt2 conditional knockout mice.

<p>(A) Schematic of the gene targeting strategy. The neomycin resistance cassette and flanked LoxP1 sequences were inserted into intron 1 for positive selection. The second LoxP2 sequences were inserted into intron 2. (B) The results of PCR genotyping. Primers 5 and 6 were used for the genotyping of floxed mice Sidt2^F/+, Sidt2^{F/+: Cre} (Sidt2^+/−) and WT (Sidt2^+/+). Primers 7 and 8 were used to differentiate Sidt2^−/+, Sidt2^−/−, Sidt2^+/+ and Sidt2^F/F, while the primers 9 and 10 were used to confirm Cre deletion. (C) RT-PCR analysis of Sidt2 mRNA (extracted from tail tissue). (D) Sidt2 mRNA detection in various tissues with β-actin loaded as an internal control. (E) Western blot analysis of Sidt2 protein in the liver of KO mice with GAPDH loaded as an internal control. (F) Appearance of mice at birth and as adults. (G) Body weight change of Sidt2^−/− mice (n = 35–50).</p

Increased insulin protein level in Sidt2^−/− mice.

<p>(A) H&E staining of islets in pancreatic sections. Bars: 1 mm. (B) The percentage of three islet types. Quantity of 636 islets from three Sidt2 −/− mice and 872 islets from <u>four</u> Sidt2 +/+ mice were determined. Total islets quantities is presented as mean ± SEM. ** P<0.01. (C) mRNA (left) and Western blot (right) analysis of insulin expression in the islets. The glucagon mRNA was also analyzed (left). * P<0.05, n = 12. (D) Immunofluorescence staining for insulin (green), glucagon (red) and DAPI (blue) in pancreatic sections. (E) Histogram showing the relative insulin expression levels calculated by the fluorescence signals in Panel D. * P<0.05, n = 4–6. (F) Count of beta cells. (G) TEM images of β-cells.</p

Blood glucose changes in Sidt2^−/− mice at different ages.

<p>(A) Random blood glucose levels in 2–16 weeks of age (n = 10–12). (B) Fasting blood glucose levels in the two groups at different age (n = 10–15, fasting overnight). (C) IPGTT at 4 weeks old (n = 8). (D) IPGTT at 8 weeks of age (n = 12). (E)AUC_{0–120 min} of the blood glucose shown in panel C and D. (F) Plasma insulin level after glucose stimulation intraperitoneally at 1.5 g/kg body weight. Data is presented as mean ± SEM (n = 7–9). All values are the means ± SEM. * P<0.05, ** P<0.01.</p

Additional file 1: Table S1. of Copy number variations in 119 Chinese children with idiopathic short stature identified by the custom genome-wide microarray

List of 1469 height-associated candidate genes analyzed by genome-wide association studies. Table S2. Summary of type III and IV CNVs. Table S3. Primers of qPCR. Figure S1. Screenshots of chromosome microarray and qPCR. (DOC 7216 kb

Primers used for amplifying exons and cDNA.

<p>Primers used for amplifying exons and cDNA.</p

Homology alignment of amino acid sequences among IDS, N-acetylgalactosamine-6S sulfatase, arylsulfatase A, and arylsulfatase B.

<p>Residues identical in at least two of four sulfatases were shaded by blue, with more identity, the color used deeper. GALNS, ASA, and ASB were the short forms of N-acetylgalactosamine-6S sulfatase, arylsulfatase A, and arylsulfatase B, respectively.</p

Additional file 1 of Late-onset cblC defect: clinical, biochemical and molecular analysis

Supplementary Material

Androgen receptor X- inactivation assay in the female patient with Hunter syndrome.

<p>An X-inactivation pattern >95∶5 was noted in the female patient (P130), while her mother (P130M) showed an pattern 60∶40. After digestion by HpaII, the female patient showed the same signal as her father (P130F) indicating that the androgen receptor gene on the allele from her father was methylated and preserved.</p

Table1_Clinical, biochemical, and molecular genetic characteristics of patients with primary carnitine deficiency identified by newborn screening in Shanghai, China.DOCX

Background: Primary carnitine deficiency (PCD) is an autosomal recessive disease caused by mutations in the SLC22A5 gene, which encodes the organic cation transporter 2 (OCTN2). Patients with PCD may be at risk of skeletal or cardiac myopathy, metabolic decompensation, and even sudden death. This study aimed to analyze the biochemical, clinical, and genetic characteristics of PCD patients identified by newborn screening (NBS) in Shanghai.Methods: Dried blood spot (DBS) samples of newborns were analyzed through tandem mass spectrometry (MS/MS) from January 2003 to December 2021. Newborns with low free carnitine (C0) levels were recalled. Mutation in the SLC22A5 gene was analyzed on suspected positive newborns with low C0 levels after recall.Results: 1,247,274 newborns were screened by MS/MS and 40 newborns were diagnosed with PCD, therefore the incidence of PCD in Shanghai was approximately 1:31,200. The mean C0 level in newborns with PCD was 5.37 ± 1.79 μmol/L before treatment and increased to 24.45 ± 10.87 μmol/L after treatment with L-carnitine. Twenty-three different variants were identified in the SLC22A5 gene, including 8 novel variants, of which c.51C>G (p.F17L) was the most frequent (27.27%, 18/66), followed by c.1400C>G (p.S467C) (25.76%, 17/66). Almost all the screened PCD patients were asymptomatic.Conclusion: NBS via MS/MS was a quick and efficient method for the early diagnosis of PCD. The incidence of PCD in Shanghai was 1:31,200. Eight novel variants were identified, which greatly expanded the variant spectrum of SLC22A5. MS/MS combined with genetic testing could effectively improve the diagnostic accuracy of PCD.</p

Image2_Clinical, biochemical, and molecular genetic characteristics of patients with primary carnitine deficiency identified by newborn screening in Shanghai, China.JPEG

Background: Primary carnitine deficiency (PCD) is an autosomal recessive disease caused by mutations in the SLC22A5 gene, which encodes the organic cation transporter 2 (OCTN2). Patients with PCD may be at risk of skeletal or cardiac myopathy, metabolic decompensation, and even sudden death. This study aimed to analyze the biochemical, clinical, and genetic characteristics of PCD patients identified by newborn screening (NBS) in Shanghai.Methods: Dried blood spot (DBS) samples of newborns were analyzed through tandem mass spectrometry (MS/MS) from January 2003 to December 2021. Newborns with low free carnitine (C0) levels were recalled. Mutation in the SLC22A5 gene was analyzed on suspected positive newborns with low C0 levels after recall.Results: 1,247,274 newborns were screened by MS/MS and 40 newborns were diagnosed with PCD, therefore the incidence of PCD in Shanghai was approximately 1:31,200. The mean C0 level in newborns with PCD was 5.37 ± 1.79 μmol/L before treatment and increased to 24.45 ± 10.87 μmol/L after treatment with L-carnitine. Twenty-three different variants were identified in the SLC22A5 gene, including 8 novel variants, of which c.51C>G (p.F17L) was the most frequent (27.27%, 18/66), followed by c.1400C>G (p.S467C) (25.76%, 17/66). Almost all the screened PCD patients were asymptomatic.Conclusion: NBS via MS/MS was a quick and efficient method for the early diagnosis of PCD. The incidence of PCD in Shanghai was 1:31,200. Eight novel variants were identified, which greatly expanded the variant spectrum of SLC22A5. MS/MS combined with genetic testing could effectively improve the diagnostic accuracy of PCD.</p