[Analysis of clinical phenotype and ACAT1 gene mutation in a family affected with beta-ketothiolase deficiency].

OBJECTIVE: To investigate the clinical phenotype and ACAT1 gene mutation in a family affected with beta-ketothiolase deficiency (BKTD). METHODS: Clinical features and laboratory test data were collected. The probands were monozygotic twin brothers. Genomic DNA was isolated from peripheral blood leukocytes obtained from the probands and their family members. Molecular genetic testing of the ACAT1 gene was carried out. RESULTS: The probands have presented with fever, vomiting and severe ketoacidosis. By arterial blood gas testing, pH was determined to be 7.164, bicarbonate was 4.0 mmol/L, and urine ketone was ++++. Urinary organic acid gas chromatography-mass spectrometry analysis showed excessive excretion of 3-hydroxybutyric acid, 2-methyl-3-hydroxybutyric acid and tiglylglycine. Increased 3-hydroxybutyrylcarnitine (C4-OH), tiglylcarnitine(C5:1) and 3-hydroxyisovalerylcarnitine (C5-OH) levels. The clinical phenotype of proband's parents were both normal, but an elder sister turned out to be an affected patient. Genetic analysis has identified two heterozygous mutations [c.622C>T(p.R208X) and c.653C>T (p.S218F)] in the proband, which were respectively detected in the mother and father. The c.653C>T (p.S218F) mutation was not found among the 100 healthy controls and has not been included in the Human Gene Mutation Database(HGMD). CONCLUSION: The primary clinical manifestations of BKTD is ketoacidosis. Urine organic acid and blood acylcarnitine analyses play an important role in the diagnosis of the disease. The compound heterozygous of ACAT1 gene mutations probably underlie the BKTD in our patient.

[Analysis of L2HGDH gene mutation in a patient with 2-hydroxyglutaric aciduria].

OBJECTIVE: To explore pathogenic mutation in a family affected with 2-hydroxyglutaric aciduria. METHODS: Exons of 3 candidate genes, including L2HGDH, D2HGDH and SLC25A1, were amplified with polymerase chain reaction and subjected to direct sequencing. RESULTS: DNA sequencing has found that the proband and his affected younger brother have both carried a heterozygous mutation c.845G>A (p.R282Q) in the exon 7 of the L2HGDH gene. The same mutation was not detected in the his sister who was healthy. Pedigree analysis has confirmed that the above mutation was inherited from the mother. No mutation was detected in exons and flanking sequences of the D2HGDH and SLC25A1 genes. CONCLUSION: Mutation of the L2HGDH gene probably underlies the 2-hydroxyglutaric aciduria in this family.

[Analysis of MUT gene mutations in a patient with isolated methylmalonic acidemia].

OBJECTIVE: To analyze the clinical features and mutation of MUT gene in a Chinese patient with isolated methylmalonic acidemia. METHODS: The clinical characteristics and laboratory tests data were collected. Genomic DNA was extracted from peripheral blood leukocytes. The 13 exons and their flanking sequences of the MUT gene were amplified with polymerase chain reaction and subjected to direct DNA sequencing. RESULTS: The patient has featured failure to thrive, lethargy, seizure, hypotonia, severe ketoacidosis and hyperammonemia. Tandem mass results showed reduction of multiple acylcarnitine. Urine organic acid testing showed pronounced increase in methylmalonate excretion. Homocysteine was normal. The patient showed no response to vitamin B12 treatment. The above results suggested that the patient had isolated methylmalonic acidemia. DNA sequencing analysis confirmed that the patient has carried two MUT gene mutations, c.755dupA and a novel mutation c.944dupT. CONCLUSION: Inherited metabolic disease screening plays an important role in the diagnosis of clinical diseases. However, to confirm the results will need gene mutation analysis.

[Analysis of PCCA and PCCB gene mutations in patients with propionic acidemia].

OBJECTIVE: To analyze PCCA and PCCB gene mutations in 10 Chinese patients with propionic acidemia(PA). METHODS: Genomic DNA was extracted from peripheral blood leukocytes. The 39 exons and flanking sequences of the PCCA and PCCB genes were amplified with polymerase chain reaction and subjected to direct DNA sequencing. RESULTS: DNA sequencing has revealed that 7 patients have carried a PCCA gene mutation, 2 patients carried PCCB gene mutation and 1 patient carried mutations in both PCCA and PCCB genes. Ten PA mutations were confirmed, including 8 affecting the PCCA gene and 2 affecting the PCCB gene. Three PCCA mutations c.245G>A, IVS15+5del5, c.1288C>T and 2 PCCB mutations c.838insC, c.1087T>C were found for the first time. CONCLUSION: Among Chinese patients with propionic acidemia patients, their genetic mutations are mainly found on the PCCA gene.

[Analysis of ornithine transcarbamylase gene mutations in three boys affected with late-onset ornithine transcarbamylase deficiency].

OBJECTIVE: To identify the types of OTC gene mutations in three male patients with late onset ornithine transcarbamylase deficiency (OTCD, MIM #311250). METHODS: Genomic DNA was extracted from peripheral blood leukocytes. The 10 exons and their flanking sequences of the OTC gene were amplified with polymerase chain reaction and subjected to direct DNA sequencing. RESULTS: Based on DNA sequence analysis, all of the three patients have carried OTC gene mutations. Patients 1 and 2 were both hemizygous for mutation c.586G> A(p.D196N). A novel mutation c.800G> C(p.S267T) were confirmed in patient 3. CONCLUSION: p.S267T mutation has affected the conserved amino acid motif of the OTC protein, and is therefore a pathogenic mutation.

[Genetic analysis of ASS1, ASL and SLC25A13 in citrullinemia patients].

OBJECTIVE: To detect potential mutations of Y9ASS1, ASL and SLC25A13 genes in four patients manifesting citrullinemia. METHODS: Genomic DNA was extracted from peripheral blood leukocytes. Exons and their flanking sequences of the three genes were amplified with polymerase chain reaction and subjected to direct DNA sequencing. RESULTS: Based on DNA sequence analysis, one case was diagnosed with argininosuccinate synthetase deficiency, and the mutation type (ASS1 gene) was c.236C>T (p.S79F) + c.431C>G (p.P144R). Two cases were diagnosed with argininosuccinic aciduria (ASL gene), and their gene mutations were c.434A>G (p.D145G) + c.1366C>T (p.R456W) and c.331C>T (p.R111W) + IVS8+2insT, respectively. A thirteen months boy who carried a heterozygous 851del4 mutation (SLC25A13 gene) was diagnosed with citrullinemia adult-onset type II. CONCLUSION: Through analysis of relevant pathogenic genes, four patients have been diagnosed.

[Clinical investigation and mutation analysis of a child with citrin deficiency complicated with purpura, convulsive seizures and methioninemia].

OBJECTIVE: To analyze the clinical features and SLC25A13 gene mutations of a child with citrin deficiency complicated with purpura, convulsive seizures and methioninemia. METHODS: The patient was subjected to physical examination and routine laboratory tests. Blood amino acids and acylcarnitines, and urine organic acids and galactose were analyzed respectively with tandem mass spectrometry and gas chromatographic mass spectrometry. SLC25A13 gene mutation screening was conducted by high resolution melt (HRM) analysis. RESULTS: The petechiae on the patient's face and platelet count (27×10(9)/L, reference range 100×10(9)/L-300×10(9)/L) supported the diagnosis of immunologic thrombocytopenic purpura (ITP). Laboratory tests found that the patient have abnormal coagulation, cardiac enzyme, liver function and liver enzymes dysfunction. Tandem mass spectrometry also found methionine to be increased (286 µmol/L, reference ranges 8-35 µmol/L). The patient did not manifest any galactosemia, citrullinemia and tyrosinemia. Analysis of SLC25A13 gene mutation found that the patient has carried IVS16ins3kb, in addition with abnormal HRM result for exon 6. Direct sequencing of exon 6 revealed a novel mutation c.495delA. The same mutation was not detected in 100 unrelated healthy controls. Further analysis of her family has confirmed that the c.495delA mutation has derived from her farther, and that the IVS16ins3kb was derived from her mother. CONCLUSION: The clinical features and metabolic spectrum of citrin deficiency can be variable. The poor prognosis and severity of clinical symptoms of the patient may be attributed to the novel c.495delA mutation.

[Analysis of clinical features and GCDH gene mutations in four patients with glutaric academia type I].

OBJECTIVE: To review clinical features of four male patients with glutaric academia type I and screen glutaryl-CoA dehydrogenase (GCDH) gene mutations. METHODS: The 4 patients underwent brain computer tomography (CT) and magnetic resonance imaging (MRI) analyses. Blood acylcarnitine and urine organic acid were analyzed with tandem mass spectrometry and gas chromatographic mass spectrometry. Genomic DNA was extracted from peripheral blood samples. The 11 exons and flanking sequences of GCDH gene were amplified with PCR and subjected to direct DNA sequencing. RESULTS: All patients have manifested macrocephaly, with head circumference measured 50 cm (14 months), 47 cm (9 months), 46 cm (5 months) and 51 cm (14 months), respectively. Imaging analyses also revealed dilation of Sylvian fissure and lateral ventricles, frontotemporal atrophy, subarachnoid space enlargement and cerebellar vermis abnormalities. All patients had elevated glutarylcarnitine (5.8 umol/L, 7.5 umol/L, 8.3 umol/L and 7.9 umol/L, respectively) and high urinary excretion of glutaric acid. Seven mutations were identified among the patients, among which c.146_149del4, IVS6-4_Ex7+4del8, c.508A>G (p.K170E), c.797T>C (p.M266T) and c.420del10 were first discovered. CONCLUSION: Macrocephaly and neurological impairment are the most prominent features of glutaric academia type I. Blood tandem mass spectrometry and urine gas chromatographic mass spectrometry analysis can facilitate the diagnosis. The results can be confirmed by analysis of GCDH gene mutations.

[Utilization of high-resolution melting analysis to screen patients with neonatal intrahepatic cholestasis caused by citrin deficiency].

OBJECTIVE: To assess the feasibility of high-resolution melting (HRM) analysis for screening patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). METHODS: Based on previous studies on SLC25A13 gene in Chinese patients with NICCD, four hotspot mutations (851del4, 1638ins23, IVS6+5G>A and IVS16ins3kb) were selected. Results of the HRM analysis was validated using 50 negative controls and 20 patients with NICCD whose genotypes were confirmed previously by direct sequencing. With the established protocol, 171 suspected patients were enrolled. Samples with abnormal melting curves were further validated by DNA sequencing. RESULTS: HRM analysis can accurately determine the genotypes of all negative controls and patients. The sensitivity and specificity of the technique reached 100% (70/70). The melting curves of samples with the same genotype were highly reproducible. In 171 suspected patients, seven NICCD patients were detected by HRM. Identified mutations have included one case of 851del4 homozygote, one case of IVS6+5G>A heterozygote, 3 cases of 851del4 heterozygotes, one case of [IVS6+5G>A]+[ 851del4] and one case of [1638ins23+IVS16ins3kb]+[1638ins23]. All mutations were subsequently confirmed by DNA sequencing. CONCLUSION: HRM analysis is a convenient, high-throughput and rapid technique for the screening of NICCD patients.

[SLC25A13 gene analysis in neonates with intrahepatic cholestasis caused by citrin deficiency].

OBJECTIVE: Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) which resulted from mutation in SLC25A13 gene can present transient intrahepatic cholestasis, low birth weight, growth retardation, hypoproteinemia and so on. This study aimed to identify the mutation type of NICCD patients by DNA sequencing. METHODS: Twenty children diagnosed as NICCD were consented to enroll in this study. PCR assays were performed to amplify the eighteen exons and its flanking sequences of SLC25A13 gene, which were defined as the upstream and downstream 50 bp from starting and ending site of the exons. Then the PCR products were purified and followed by automated DNA sequencing. The IVS16ins3kb mutation was detected by nested PCR and RT-PCR. RESULTS: Seven genetic variations of SLC25A13, termed as 851del4, 1638ins23, IVS16ins3kb, IVS6+5G>A, c.775C>T (p.Q259X), c.1505C>T (p.P502L) and c.1311C>T (p.C437C), were identified in the subjects, of which c.775C>T (p.Q259X), c.1505C>T (p.P502L) and c.1311C>T (p.C437C) were reported for the first time in NICCD patients. And a compound mutation ofï¼»1638ins23+IVS16ins3kbï¼½was also identified. In 20 patients with NICCD, 6 patients were 851del4 homozygotes, 7 patients were compound heterozygotes, and 7 patients were heterozygotes of single mutation. 851del4 was the major mutation type (64%), followed by 1638ins23 (15%), IVS16ins3kb (12%) and IVS6+5G>A (6%). CONCLUSIONS: 851del4 is the major mutation type in Chinese patients with NICCD.