[Analysis of IVD gene variants in four children with isovalerate acidemia].

OBJECTIVE: To detect variants of IVD gene among 4 neonates with suspected isovalerate acidemia in order to provide a guidance for clinical treatment. METHODS: 111 986 newborns and 7461 hospitalized children with suspected metabolic disorders were screened for acyl carnitine by tandem mass spectrometry. Those showing a significant increase in serum isovaleryl carnitine (C5) were analyzed for urinary organic acid and variants of the IVD gene. RESULTS: Four cases of isovalerate acidemia were detected, which included 2 asymptomatic newborns (0.018‰, 2/111 986) and 2 children suspected for metabolic genetic diseases (0.268‰, 2/7461). The formers had no obvious clinical symptoms. Analysis of acyl carnitine has suggested a significant increase in C5, and urinary organic acid analysis has shown an increase in isovaleryl glycine and 3-hydroxyisovalerate. Laboratory tests of the two hospitalized children revealed high blood ammonia, hyperglycemia, decreased red blood cells, white blood cells, platelets and metabolic acidosis. The main clinical manifestations have included sweaty foot-like odor, feeding difficulty, confusion, drowsiness, and coma. Eight variants (5 types) were detected, which included c.158G>A (p.Arg53His), c.214G>A (p.Asp72Asn), c.548C>T (p.Ala183Val), c.757A>G (p.Thr253Ala) and 1208A>G (p.Tyr403Cys). Among these, c.548C>T and c.757A>G were unreported previously. None of the variants was detected by next generation sequencing of 2095 healthy newborns, and all variants were predicted to be likely pathogenic based on the guidelines from the American College of Medical Genetics and Genomics. CONCLUSION: The incidence of isovalerate acidemia in Liuzhou area is quite high. Screening of metabolic genetic diseases is therefore recommended for newborns with abnormal metabolism. The discovery of novel variants has enriched the mutational spectrum of the IVD gene.

[Analysis of PLA2G6 gene variant in a family affected with infantile neuroaxonal dystrophy].

OBJECTIVE: To identify potential variant in a child diagnosed as infantile neuroaxonal dystrophy. METHODS: Genomic DNA was extracted from peripheral blood samples from the patient and his parents and subjected to next generation sequencing. Suspected variant was verified by PCR and Sanger sequencing. Pathogenicity of the mutation was predicted by using bioinformatic software including SIFT and PolyPhen-2. RESULTS: The child was found to carry compound heterozygous variations c.668C>A (p.Pro223Gln) and c.2266C>T (p.Gln756Ter) of the PLA2G6 gene, which were respectively inherited from his father and mother. c.2266C>T has changed codon 756 (glutamine) into a stop codon, resulting premature termination of peptide chain synthesis. c.2266C>T has not been reported previously and was predicted to be harmful. CONCLUSION: The compound variants of c.668C>A (p.Pro223Gln) and c.2266C>T (p.Gln756Ter) of the PLA2G6 gene probably underlies the disease in the child. Above finding has enriched the variant spectrum of the PLA2G6 gene.

[Analysis of TRPM6 gene variant in a pedigree affected with hypocalcemia secondary to hypomagnesemia].

OBJECTIVE: To explore the molecular pathogenesis for a pedigree affected with hypocalcemia secondary to hypomagnesemia. METHODS: Sanger sequencing was used to detect potential variant of the TRPM6 gene in the patient and their parents. RESULTS: The results showed that the patient has carried novel homozygous c.3311C>T (p.Pro1104Leu) variant of the TRMP6 gene, for which both of his parents were heterozygous carriers. Analysis of protein functions using software predicted high risk of pathogenicity. CONCLUSION: The homozygous c.3311C>T (p.Pro1104Leu) variant of the TRPM6 gene probably underlies the disease in this patient.

[Analysis of CGDH gene variants and clinical features in three patients with glutaric aciduria type â ].

OBJECTIVE: To screen for potential variants of GCDH gene in 3 patients clinically diagnosed as glutaric aciduria type Ⅰ. METHODS: GCDH gene variants was detected by Sanger sequencing among the three children and their family members. RESULTS: Sanger sequencing showed that patient 1 carried compound heterozygosity variants of c.532G>A (p.Gly178Arg) and c.655G>A (p.Ala219Thr) of the GCDH gene, while his father and mother respectively carried heterozygous c.532G>A(p.Gly178Arg) and c.655G>A (p.Ala219Thr) variants. Patient 2 carried c.532G>A (p.Gly178Arg) and a novel c.1060G>T (p.Gly354Cys) compound heterozygous variant, while his father and mother respectively carried heterozygous c.532G>A (p.Gly178Arg) and c.1060G>T (p.Gly354Cys) variant. Patient 3 carried homozygous c.532G>A (p.Gly178Arg) variant of the GCDH gene, for which both of his parents were heterozygous carriers. CONCLUSION: The GCDH gene variant probably underlie the glutaric aciduria type Ⅰ among the 3 patients. Identifcation of the novel variant has enriched the spectrum of GCDH gene variants.

[Tandem mass spectrometry analysis and genetic diagnosis of neonates with fatty acid oxidation disorders in central and northern regions of Guangxi].

OBJECTIVE: To determine the incidence and mutational types of fatty acid oxidation disorders (FAOD) in central-northern region of Guangxi. METHODS: A total of 62 953 neonates were screened for FAOD during December 2012 and December 2017. Acyl-carnitine profiling of neonatal blood sample was performed by tandem mass spectrometry using dry blood spots on a filter paper. The diagnosis of FAOD was confirmed by organic acid profiling of urea and genetic testing. RESULTS: Eighteen cases of FAOD were diagnosed among the 62 953 neonates. Among these, primary carnitine deficiency (PCD) was the most common type (n=13), which was followed by short-chain acyl-CoA dehydrogenase deficiency (SCADD) (n=2), medium-chain acyl-CoA dehydrogenase deficiency (MCADD) (n=1), multiple acyl-CoA dehydrogenase deficiency (MADD) (n=1), and carnitine palmitoyltransferase II deficiency (CPT II D) (n=1). Genetic testing has revealed two previously unreported variants, i.e., c.337G to A (p.Gly113Arg) of ACADS gene and c.737G TO T (p.Gly246Val) of ETFA gene. CONCLUSION: PCD is the most common FAOD in central-northern Guangxi. Tandem mass spectrometry combined with genetic testing may facilitate early diagnosis of FAOD.

[SLC22A5 gene mutation analysis and prenatal diagnosis for a family with primary carnitine deficiency].

OBJECTIVE: To carry out mutation analysis and prenatal diagnosis for a family affected with primary carnitine deficiency. METHODS: Genomic DNA of the proband was extracted from peripheral blood sample 10 days after birth. The 10 exons and intron/exon boundaries of the SLC22A5 gene were subjected to PCR amplification and Sanger sequencing. The proband's mother was pregnant again two years after his birth. Fetal DNA was extracted from amniocytes and subjected to PCR and Sanger sequencing. RESULTS: Tandem mass spectrometric analysis of the proband revealed low level of plasma-free carnitine whilst organic acids in urine was normal. Compound heterozygous SLC22A5 mutations c.1195C>T (inherited from his father) and c.517delC (inherited from his mother) were detected in the proband. Prenatal diagnosis has detected no mutation in the fetus. The plasma-free carnitine was normal after birth. CONCLUSION: Appropriate genetic testing and prenatal diagnosis can prevent further child with carnitine deficiency. The identification of c.517delC, a novel mutation, enriched the spectrum of SLC22A5 mutations.

[Clinical and genetic analysis of a child with Noonan syndrome].

OBJECTIVE: To identify potential mutation in a child clinically diagnosed as Noonan syndrome and to provide genetic counseling and prenatal diagnosis for his family. METHODS: Genomic DNA was extracted from peripheral blood samples of the patient and his parents, and amniotic fluid was taken from the mother during the second trimester. Next generation sequencing (NGS) was used to screen potential mutations from genomic DNA. Suspected mutation was verified by Sanger sequencing. RESULTS: A heterozygous c.4A>G (p.Ser2Gly) mutation of the SHOC2 gene was identified in the patient but not among other family members including the fetus. CONCLUSION: The Noonan syndrome is probably caused by the c.4A>G mutation of the SHOC2 gene. NGS is helpful for the diagnosis of complicated genetic diseases. SHOC2 gene mutation screening is recommended for patient suspected for Noonan syndrome.

[Analysis of P gene variations among fourteen patients with oculocutaneous albinism type II].

OBJECTIVE: To analyze variations of TYR and P genes among 14 patients with clinically diagnosed oculocutaneous albinism. METHODS: Potential variations of the TYR and P genes were detected by Sanger sequencing. Novel variations were predicted with bioinformatics software including SIFT and PolyPhen-2. RESULTS: No variation was found in the TYR gene, while 9 types of variations were found in the P gene among the 14 patients, which included c.803-3C>G (7/26), c.1327G>A (p.Val443Ile) (5/26), c.632C>T (p.Pro211Leu) (4/26), c.1832T>C (p.Leu611Pro) (3/26), c.1349C>A (p.Thr450Lys) (2/26), c.2363C>T (p.Ser788Leu) (2/26), c.2228C>T (p.Pro743Leu) (1/26), c.1525A>G (p.Thr509Ala) (1/26), and c.1349C>T (p.Thr450Met) (1/26). Only 1 heterozygous variation was detected in 2 families. c.2363C>T (p.Ser788Leu), c.1832T>C (p.Leu611Pro) and c.1525A>G (p.Thr509Ala) were not reported previously and predicted as "harmful" to the protein function. CONCLUSION: The main type of ocular albinism is oculocutaneous albinism type II in Liuzhou region, where the most common variations of the P gene were c.803-3C>G and c.1327G>A (p.Val443Ile). Above finding has enriched the variation spectrum of the P gene.

[Screening for spinal muscular atrophy mutation carriers among 4931 pregnant women from Liuzhou region of Guangxi].

OBJECTIVE: To screen for carriers of SMN1 gene mutation, which underlies spinal muscular atrophy (SMA), in 4931 pregnant women from Liuzhou region of Guangxi, and to determine the carrier rate. METHODS: Combined denaturing high-performance liquid chromatography (DHPLC) and multiple PCR techniques were used to detect the copy number of SMN1 gene. The carrier frequency was calculated. The spouse of the carrier was also screened, and prenatal diagnosis was provided to the couples who were both positive. RESULTS: Among the 4931 pregnant women, 61 were found to harbor only one copy of the SMN1 gene, which yielded a carrier rate of 1.2%. Subsequent testing has identified 1 fetus carrying homozygous deletions of the SMN1 gene. CONCLUSION: The carrier rate of SMA mutation in Liuzhou region is slightly lower than that of other regions of southern China. DHPLC can effectively screen the carriers of SMA mutation and provide a basis for genetic counseling and prenatal diagnosis.

[Screening indices and their cut-off values for full-term neonates carrying ß-thalassemia gene].

OBJECTIVE: To investigate the screening indices and their cut-off values for full-term neonates carrying ß-thalassemia gene. METHODS: A retrospective analysis was performed for the clinical data of 1 193 full-term neonates who underwent ß-thalassemia screening (hemoglobin analysis with dried blood spots on neonatal heel blood filter paper and mutation detection of 17 ß-globin genes). A multivariate logistic regression analysis was used to investigate the association between screening indices and ß-thalassemia gene, and the receiver operating characteristic (ROC) curve was used to analyze the value of screening indices in determining the presence or absence of ß-thalassemia gene. RESULTS: Of the 1 193 neonates, 638 carried ß-thalassemia gene. Of the 1 193 neonates, 637 (53.39%) had no HbA2, among whom 310 carried ß-thalassemia gene and 327 did not carry this gene; 556 (46.61%) had HbA2, among whom 328 carried ß-thalassemia gene and 228 did not carry this gene. As for the neonates without HbA2, the ß-thalassemia gene group had a significantly lower HbA level and a significantly higher HbF level than the ß-thalassemia gene-negative group (P<0.01). As for the neonates with HbA2, the ß-thalassemia gene group had a significantly lower HbA level and significantly higher HbF and HbA2/HbA ratio than the ß-thalassemia gene-negative group (P<0.01). In the neonates without HbA2, HbA, gestational age, and HbA combined with gestational age had an area under the ROC curve (AUC) of 0.865, 0.515, and 0.870, respectively, in determining the presence or absence of ß-thalassemia gene (P<0.01), and HbA and HbA combined with gestational age had a similar AUC and a certain diagnostic value. In the neonates with HbA2, HbA, HbA2/HbA ratio, and HbA combined with HbA2/HbA ratio had an AUC of 0.943, 0.885, and 0.978, respectively, in determining the presence or absence of ß-thalassemia gene. The HbA combined with HbA2/HbA ratio had the largest AUC. In the neonates without HbA2, HbA had the largest AUC in determining the presence or absence of ß-thalassemia gene at the cut-off value of 11.6%, with a sensitivity of 85.81% and a specificity of 79.82%. In the neonates with HbA2, an HbA of 16.1%-22.0% and an HbA2/HbA ratio of >1.4 had the largest AUC in determining the presence or absence of ß-thalassemia gene, with a sensitivity of 91.38% and a specificity of 91.89%. CONCLUSIONS: HbA and HbA2/HbA ratio are effective indices for screening out full-term neonates carrying ß-thalassemia gene.

[Clinical and cytogenetic study in a child with de novo chromosome 9 abnormality].

This study aimed to analyze the clinical phenotype of chromosome 9p deletion or duplication and its relationship with karyotype. A patient, female, aged 6 months, visited the hospital due to motor developmental delay. Karyotype analysis identified abnormalities of chromosome 9 short arm, and high-throughput sequencing found 9p24.3-9p23 deletion and 9p23-9p13.1 duplication. Her parents had a normal karyotype. Karyotype analysis combined with high-throughput sequencing is of great significance for improving the efficiency of etiological diagnosis in children with motor developmental delay or multiple congenital deformities and mental retardation.

[Genetic diagnosis of 10 neonates with primary carnitine deficiency].

OBJECTIVE: To study the gene mutation profile of primary carnitine deficiency (PCD) in neonates, and to provide a theoretical basis for early diagnosis and treatment, genetic counseling, and prenatal diagnosis of PCD. METHODS: Acylcarnitine profile analysis was performed by tandem mass spectrometry using 34 167 dry blood spots on filter paper. The SLC22A5 gene was sequenced and analyzed in neonates with free carnitine (C0) levels lower than 10 µmol/L as well as their parents. RESULTS: In the acylcarnitine profile analysis, a C0 level lower than 10 µmol/L was found in 10 neonates, but C0 level was not reduced in their mothers. The 10 neonates had 10 types of mutations at 20 different sites in the SLC22A5 gene, which included 4 previously unreported mutations: c.976C>T, c.919delG, c.517delC, and c.338G>A. Bioinformatics analysis showed that the four new mutations were associated with a risk of high pathogenicity. CONCLUSIONS: Tandem mass spectrometry combined with SLC22A5 gene sequencing may be useful for the early diagnosis of PCD. Identification of new mutations enriches the SLC22A5 gene mutation profile.

[An analysis of clinical characteristics and gene mutation in two patients with medium- and short-chain acyl-CoA dehydrogenase deficiency].

Medium- and short-chain acyl-CoA dehydrogenase deficiency is a disorder of fatty acid ß-oxidation. Gene mutation prevents medium- and short-chain fatty acids from entry into mitochondria for oxidation, which leads to multiple organ dysfunction. In this study, serum acylcarnitines and the organic acid profile in urea were analyzed in two children whose clinical symptoms were hypoglycemia and metabolic acidosis. Moreover, gene mutations in the two children and their parents were evaluated. One of the patients was a 3-day-old male who was admitted to the hospital due to neonatal asphyxia, sucking weakness, and sleepiness. The serum acylcarnitine profile showed increases in medium-chain acylcarnitines (C6-C10), particularly in C8, which showed a concentration of 3.52 µmol/L (reference value: 0.02-0.2 µmol/L). The analysis of organic acids in urea gave a normal result. Sanger sequencing revealed a reported c.580A>G (p.Asn194Asp) homozygous mutation at exon 7 of the ACADM gene. The other patient was a 3-month-old female who was admitted to the hospital due to cough and recurrent fever for around 10 days. The serum acylcarnitine profile showed an increase in serum C4 level, which was 1.66 µmol/L (reference value: 0.06-0.6 µmol/L). The analysis of organic acids in urea showed an increase in the level of ethyl malonic acid, which was 55.9 (reference value: 0-6.2). Sanger sequencing revealed a reported c.625G>A (p.Gly209Ser) homozygous mutation in the ACADS gene. This study indicates that screening tests for genetic metabolic diseases are recommended for children who have unexplained metabolic acidosis and hypoglycemia. Genetic analyses of the ACADM and ACADS genes are helpful for the diagnosis of medium- and short-chain acyl-CoA dehydrogenase deficiency.

[Pancytopenia and metabolic decompensation in a neonate].

A 9-day-old male patient was admitted to the hospital because of cough, anhelation, feeding difficulty and lethargy. The diagnostic examinations indicated pulmonary infection, severe metabolic acidosis, hyperglycemia, hyperammonemia and pancytopenia in the patient. Blood and urine screening and isovaleryl-CoA dehydrogenase (IVD) gene detection for inherited metabolic diseases were performed to clarify the etiology. Tandem mass spectrometric screening for blood showed an elevated isovalerylcarnitine (C5) level. The organic acid analysis of urine by gas chromatography-mass spectrometry showed significantly increased levels in isovaleryl glycine and 3-hydroxyisovaleric acid. Homozygous mutations (c.1208A>G, p.Tyr403Cys) in the IVD gene were identified in the patient. His parents were heterozygous carriers. After the treatment with low-leucine diets and L-carnitine for 3 days, the patient showed a significant improvement in symptoms, but he died one week later. It is concluded that the neonates with pneumonia and metabolic decompensation of unknown etiology should be screened for genetic metabolic disease.

[CPT2 gene mutation analysis and prenatal diagnosis in a family with carnitine palmitoyltransferase II deficiency].

This study aimed to identify the type of carnitine palmitoyltransferase 2 (CPT2) gene mutation in the child with carnitine palmitoyltransferase II (CPT II) deficiency and her parents and to provide the genetic counseling and prenatal diagnosis for the family members. As the proband, a 3-month-old female baby was admitted to the hospital due to fever which had lasted for 8 hours. Tandem mass spectrometric analysis for blood showed an elevated plasma level of acylcarnitine, which suggested CPT II deficiency. The genomic DNA was extracted from peripheral blood of the patient and her parents. Five exon coding regions and some intron regions at the exon/intron boundaries of the CPT2 gene were analyzed by PCR and Sanger sequencing. Amniotic fluid was taken from the mother during the second trimester, and DNA was extracted to analyze the type of CPT2 gene mutation. Sanger sequencing results showed that two mutations were identified in the CPT2 gene of the proband: c.886C>T (p.R296X) and c.1148T>A (p.F383Y), which were inherited from the parents; the second child of the mother inherited the mutation of c.886C>T (p.R296X) and showed normal acylcarnitine spectrum and normal development after birth. It is concluded that the analysis of CPT2 gene mutations in the family suggested that the proband died of CPT II deficiency and that the identification of the mutations was helpful in prenatal diagnosis in the second pregnancy.

[Molecular diagnosis for a patient with Kennedy disease].

OBJECTIVE: To screen for potential mutations of androgen receptor (AR) gene in a patient clinically diagnosed as Kennedy disease. METHODS: Polyglutamine expansion (PQE) induced by a duplication of CAG trinucleotide tandem-repeat in exon 1 of the AR gene was detected with PCR and T-clone sequencing. RESULTS: Compared with the number of CAG repeat of 22 in the normal allele, the number of CAG repeats has increased to 45 in the mutant allele carried by the patient. This has fit with the diagnostic criteria for Kennedy disease. CONCLUSION: A mutation of PQE has been detected in the patient with Kennedy disease. Detection of PQE in AR gene can be used as reliable method to identify the Kennedy disease.

Comparison of the Antianhedonic Effects of Repeated-dose Intravenous Ketamine in Older and Younger Adults with Major Depressive Episode.

OBJECTIVES: Growing evidence suggests that repeated-dose intravenous ketamine in patients with depression had rapid antianhedonic effects. However, a comparison of the antianhedonic effects of repeated-dose intravenous ketamine between younger adults and older depressed patients has not been examined. METHODS: To the best of my knowledge, this study with a total of 135 patients with major depressive episodes (MDE) is the first to compare the antianhedonic effects between younger adult (n = 116) and older (n = 19) depressed patients receiving six ketamine infusions (0.5 mg/kg over 40 min). Montgomery- Åsberg Depression Rating Scale (MADRS) was applied in this study to evaluate the clinical symptoms, and MADRS anhedonia item scoring was used to evaluate anhedonia symptoms. RESULTS: Patients received six open-label intravenous infusions of ketamine for 12 days. MADRS anhedonia subscale scores decreased in both younger (3.3, 95% CI = 2.5-4.1, p < 0.05) and older (2.8, 95% CI = 1.1-4.6, p < 0.05) MDE patients at 4h after the first infusion compared to baseline scores and the reduction was maintained over the subsequent infusion period in both groups (all Ps < 0.05). Younger MDE patients had lower MADRS anhedonia subscale scores on day 26 compared with older patients (P = 0.02). Compared with younger adult MDE patients, older patients had a lower antianhedonic response (51.7% [95% CI = 42.5%-61.0%] versus 31.6% [95% CI = 8.6%-54.6%)] and remission (24.1% [95% CI = 16.2%-32.0%] versus 0%). CONCLUSION: This study indicates that repeated-dose intravenous ketamine administration induces rapid and robust antianhedonic effects in older MDE patients. However, older MDE patients displayed less response to ketamine than younger adult MDE patients.

Improved Genetic Characterization of Congenital Adrenal Hyperplasia by Long-Read Sequencing Compared with Multiplex Ligation-Dependent Probe Amplification Plus Sanger Sequencing.

Genetic analysis of congenital adrenal hyperplasia (CAH) has been challenging because of high homology between CYP21A2 and its pseudogene CYP21A1P. This study aimed to evaluate the clinical utility of long-read sequencing (LRS) in diagnosis of CAH attributable to 21-hydroxylase deficiency by comparing with multiplex ligation-dependent probe amplification plus Sanger sequencing. In this retrospective study, 69 samples, including 49 probands from 47 families with high-risk of CAH, were enrolled and blindly subjected to detection of CAH by LRS. The genotype results were compared with control methods, and discordant samples were validated by additional Sanger sequencing. LRS successfully identified biallelic variants of CYP21A2 in the 39 probands diagnosed as having CAH. The remaining 10 probands were not patients with CAH. Additionally, LRS directly identified two pathogenic single-nucleotide variations (SNVs; c.293-13C/A>G and c.955C>T) in the presence of interference caused by nearby insertions/deletions (indels). The cis-trans configuration of two or more SNVs and indels identified in 18 samples was directly determined by LRS without family analysis. Eight CYP21A1P/A2 or TNXA/B deletion chimeras, composed of five subtypes, were identified; and the junction sites were precisely determined. Moreover, LRS determined the exact genotype in two probands who had three heterozygous SNVs/indels and duplication, which could not be clarified by control methods. These findings highlight that LRS could assist in more accurate genotype imputation and more precise CAH diagnosis.

[Analysis of SCA3/MJD3 gene mutation and genetic polymorphism in a guangxi family with spinocerebellar ataxia 3].

Autosomal dominant cerebellar ataxias (ADCAs) comprise a group of genetically heterogeneous neurodegenerative disorders among which spinocerebellar ataxia type 3 (SCA3) represents the most common form of SCAs worldwide. The fragments of SCA3/MJD gene,which is the member of family GXPL1,were amplified by polymerase chain reaction (PCR). The PCR products of SCA3/MJD gene were detected with capillary electrophoresis (CE) and sequencing to evaluate the size of CAG repeats, feature in the transmission and the mutation in the family with SCA3 in Guangxi province. The results showed that the exon 10 of the SCA3/MJD gene contains 64-71 CAG repeats in all of the affected individuals and three asymptomatic carriers of the family. The number of the CAG repeats during transmission in the normal individuals carrying CGG allele remains consistent, suggesting that CGG allele could have no effect on intergenerational stability of CAG repeats in normal individuals. In addition, two novel point mutations were identified: IVS9-113 T > C in the intronic region and a missense mutation 220 G > A (Glu > Gly) in the encoding region. These two novel point mutations have not been reported and the effect of the mutations on the phenotype of SCA3 is not clear.

The Effects of Dietary Protein Level on the Growth Performance, Body Composition, Intestinal Digestion and Microbiota of Litopenaeus vannamei Fed Chlorella sorokiniana as the Main Protein Source.

This study investigated the effect of dietary protein levels on Litopenaeus vannamei. Five isolipid diets with protein levels of 32%, 36%, 40%, 44% and 48% were prepared using C. sorokiniana as the main protein source. L. vannamei (initial body weight 0.83 ± 0.02 g) were fed these five diets for 8 weeks and referred to as the CHL32, CHL36, CHL40, CHL44 and CHL48 groups, respectively. When the feeding trial was finished, the growth performance, body composition, intestinal digestion and microbiota of L. vannamei were studied. The results showed that the maximum weight gain rate (WGR) of L. vannamei was in the CHL40 group while the lowest feed conversion ratio (FCR) was in the CHL48 group. According to the regression analysis using WGR as the evaluation index, the best growth performance of L. vannamei was obtained when the dietary protein level was 40.81%. The crude protein content of whole shrimp showed an increasing and then decreasing trend with increasing dietary protein levels. Furthermore, the L. vannamei muscle amino acid composition was relatively stable and, to some extent, independent of dietary protein levels. Trypsin, lipase and amylase (AMS) activity increased and then decreased with increasing dietary protein levels and, significantly, peaked in the CHL44 group. Analysis of the alpha diversity of the intestinal microbiota showed that the Chao1 index peaked in the CHL40 group and was significantly lower in the CHL48 group. Additionally, the relative abundance of pathogenic bacteria decreased significantly while the relative abundance of beneficial bacteria increased significantly in the intestine of L. vannamei as the dietary protein levels increased. The functional prediction of the intestinal microbiota revealed that dietary protein levels may influence the growth of L. vannamei by regulating various metabolic activities, and the highest WGR in the CHL40 group may have been related to the significant enrichment of nicotinate and nicotinamide metabolism and biotin metabolism functions. In summary, the optimal protein requirement for L. vannamei was around 40% when C. sorokiniana was used as the primary protein source. Too high or too low dietary protein levels could adversely affect shrimp body composition, intestinal digestion and microbiota.