Summary of statistical values of OPLS-DA with different scaling methods for data obtained from LC-MS analyses^a.

a<p>Including the different cumulated modeled variations in X [R²X(cum)] and Y [R²Y(cum)] matrix on spectral datasets and predictability of the model [Q²(cum)].</p

Comparison of clinical data among four groups.

<p>¹χ2-test.</p>2<p>one-way ANOVA followed by a LSD Test.</p>a<p>Comparison vs normal group p<0.05.</p>b<p>Comparison vs hyperbilirubinemia and normal group p<0.05.</p>c<p>Comparison vs NHS group, hyperbilirubinemia and normal group p<0.05.</p

VIP value of the potential metabolites in the differences of NHS and BA^a.

a<p>VIP: The variable importance for projection.</p

OPLS-DA score scatter plots obtained from of LCMS analysis of samples.

<p>A. OPLS-DA score plots of BA and normal; B. OPLS-DA score plots of with NHS and normal; C. OPLS-DA score plots of BA and NHS; D. OPLS-DA score plots of hyperbilirubinemia and normal. The fig. showing that the two populations are well separated respectively.</p

The amino acid and acylcarnitine with a VIP value above 1.0 were identified as markers to discriminate the two comparing group.

<p>The Variables were aligned in descending order of their VIP values. From model A and model B, we can obtain the identified metabolites of BA and NHS. From model C, we can verify these metabolites obtained above can distinguish BA from NHS. From model D, we can exclude the interference of metabolites in hyperbilirubinemia infants.</p

A typical LC-MS/MS spectrum of blood spots from patients.

<p>Blood samples were prepared and mass spectrometric analysis of individual sample was performed as described in “Methods” in details. Panel A shows a mass spectrum was acquired in the neutral loss scan mode. The scan was range from m/z 140 to m/z 280. It can detect most of the amino acids. Panel B shows a mass spectrum was acquired in the multiple reaction monitor mode. It was mainly used to detect Gly, Orn, Arg, Cit and its internal standard. Panel C shows a mass spectrum was acquired in precursor ion scanning mode. It used to detect the acylcarnitine in the sample.</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

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

Primers used for amplifying exons and cDNA.

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

Image1_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