Case Report: Long-term follow-up of desert hedgehog variant caused 46, XY gonadal dysgenesis with multiple complications in a Chinese child.

Background: Desert hedgehog (DHH), as a member of the Hedgehog (HH) family, is mainly involved in testicular development and peripheral nerve sheath formation. A DHH variant has been identified in patients with 46, XY gonadal dysgenesis (46, XY GD) with or without neuropathy, but few reports mention the involvement of other complications. Case presentation: Here, we report a Chinese female patient who was hospitalized at 14.3 years old due to slow breast development for more than 1 year. She had a female genitalia phenotype and breast development started at 13 years old but progressed slowly. She was not yet menarche on admission, and she had intermittent muscle cramps in her hands and feet. Her karyotype analysis was 46, XY and the SRY gene was positive. Surgical exploration revealed no uterus or ovaries, and the pathology of bilateral gonads was dysplastic testis tissue, which was consistent with partial gonadal dysgenesis (PGD). Genetic analysis identified a homozygous pathogenic variant in DHH exon 3 (c.1027T>C, p. Cys343Arg). During the 6-year follow-up, she received estrogen replacement therapy, resulting in breast development progression without gender dysphoria. However, her peripheral neuropathy became more obvious, and a nerve conduction study (NCS) indicated decreased nerve conduction velocity and action potential. In addition, she also suffered complications such as obesity, insulin resistance, fatty liver, and gastric ulcers. Conclusion: In the present study, we reported a case of 46, XY GD with minifascicular neuropathy caused by a DHH homozygous variant, and we summarized the reported cases worldwide. For the first time in such patients, we showed a comparison of NCS changes with age as well as the presence of multiple complications not previously reported.

Ubiquitination and deubiquitination of 4E-T regulate neural progenitor cell maintenance and neurogenesis by controlling P-body formation.

During embryogenesis, neural stem/progenitor cells (NPCs) proliferate and differentiate to form brain tissues. Here, we show that in the developing murine cerebral cortex, the balance between the NPC maintenance and differentiation is coordinated by ubiquitin signals that control the formation of processing bodies (P-bodies), cytoplasmic membraneless organelles critical for cell state regulation. We find that the deubiquitinase Otud4 and the E3 ligase Trim56 counter-regulate the ubiquitination status of a core P-body protein 4E-T to orchestrate the assembly of P-bodies in NPCs. Aberrant induction of 4E-T ubiquitination promotes P-body assembly in NPCs and causes a delay in their cell cycle progression and differentiation. In contrast, loss of 4E-T ubiquitination abrogates P-bodies and results in premature neurogenesis. Thus, our results reveal a critical role of ubiquitin-dependent regulation of P-body formation in NPC maintenance and neurogenesis during brain development.

[Gender difference in clinical manifestations of KBG syndrome due to variants of ANKRD11 gene].

OBJECTIVE: To explore gender difference in the clinical manifestations of two children with Keishi-Bukuryo-Gan syndrome (KBGS). METHODS: Clinical manifestations of the two children were reviewed. Genetic testing was carried out through next generation sequencing (NGS). Treatment was summarized, and the prognosis was followed up. RESULTS: Both children showed particular appearance including megatooth, abnormal hair distribution, hands' abnormality and language development delay. NGS revealed that both children have carried pathogenic variants of the ANKRD11 gene (c.1903_1907del and c.4911delT), which resulted in shifting of amino acid sequences starting from the Lysine and Proline at positions 635 and 1638, respectively. The female patient exhibited central precocious puberty. Her height has increased by 13 cm, and sex characteristics has retracted after treatment with leuprorelin for 23 months and recombinant human growth hormone for 1 month. CONCLUSION: Comparison of the two cases with different genders and summary of previously reported cases found that male KBGS patients have more obvious dysmorphisms such as triangular face, synophrys, ocular hypertelorism and vertebral body abnormality, with higher morbidity of epilepsy, mental retardation, autism, congenital heart disease, immune thrombocytopenia and other complications. KBGS is an autosomal dominant disease featuring more evident peculiar appearance and global development delay. Male patients often have multi-system involvement, and multidisciplinary cooperation is required for early recognition of particular features in order to improve the prognosis.

Nucleocytoplasmic transport of the RNA-binding protein CELF2 regulates neural stem cell fates.

The development of the cerebral cortex requires balanced expansion and differentiation of neural stem/progenitor cells (NPCs), which rely on precise regulation of gene expression. Because NPCs often exhibit transcriptional priming of cell-fate-determination genes, the ultimate output of these genes for fate decisions must be carefully controlled in a timely fashion at the post-transcriptional level, but how that is achieved is poorly understood. Here, we report that de novo missense variants in an RNA-binding protein CELF2 cause human cortical malformations and perturb NPC fate decisions in mice by disrupting CELF2 nucleocytoplasmic transport. In self-renewing NPCs, CELF2 resides in the cytoplasm, where it represses mRNAs encoding cell fate regulators and neurodevelopmental disorder-related factors. The translocation of CELF2 into the nucleus releases mRNA for translation and thereby triggers NPC differentiation. Our results reveal that CELF2 translocation between subcellular compartments orchestrates mRNA at the translational level to instruct cell fates in cortical development.

[Correlation between variants of CYP21A2 gene promoter region and nonclassical 21-hydroxylase deficiency].

OBJECTIVE: To summarize the clinical characteristics of two children with nonclassical 21 hydroxylase deficiency (NC-21OHD) due to variants of CYP21A2 gene promoter region. METHODS: Clinical characteristics and the results of genetic testing were reviewed. RESULTS: The main clinical manifestations of the two children included precocious puberty with poor bone age/progression control and menstrual disorder with hirsutism. Patient 1 had compound heterozygous variants for -126C>T, -113G>A, -110T>C and p.I173N; her mother was heterozygous for -126C>T, -113G>A and -110T>C, and her father was heterozygous for p.I173N. Patient 2 had compound heterozygous variants for -126C>T, -113G>A and p.I2G, whose mother was heterozygous for -126C>T and -113G>A, and father was heterozygous for p.I2G. CONCLUSION: Diagnosis of NC-21OHD should be considered for children with hirsutism, menstrual disorder and poor bone age/progression control. The promoter region of CYP21A2 gene should be analyzed when no variant is detected in its coding regions.

Long-term follow-up in a Chinese child with congenital lipoid adrenal hyperplasia due to a StAR gene mutation.

BACKGROUND: Congenital lipoid adrenal hyperplasia (CLAH) is an extremely rare and the most severe form of congenital adrenal hyperplasia. Typical features include disorder of sex development, early-onset adrenal crisis and enlarged adrenal glands with fatty accumulation. CASE PRESENTATION: We report a case of CLAH caused by mutations in the steroidogenic acute regulatory protein (StAR) gene. The patient had typical early-onset adrenal crisis at 2 months of age. She had normal-appearing female genitalia and a karyotype of 46, XY. The serum cortisol and adrenal steroids levels were always nearly undetectable, but the adrenocorticotropic hormone levels were extremely high. Genetic analysis revealed compound heterozygous mutations at c. 229C > T (p.Q77X) in exon 3 and c. 722C > T (p.Q258X) in exon 7 of the StAR gene. The former mutation was previously detected in only two other Chinese CLAH patients. Both mutations cause truncation of the StAR protein. The case reported here appears to be a classic example of CLAH with very small adrenal glands and is the second reported CLAH case with small adrenal glands thus far. In a 15-year follow-up, the patient's height was approximately average for females before age 4 and fell to - 1 SDS at 10 years of age. Her bone age was similar to her chronological age from age 4 to age 15 years. CONCLUSIONS: In conclusion, this is a classic case of CLAH with exceptionally small adrenal glands. Q77X mutation seems to be more common in Chinese CLAH patients. Additionally, this is the first report of the growth pattern associated with CLAH after a 15-year follow-up.

[Clinical features and genetic analysis of a case with carnitine palmitoyltransferase 1A deficiency].

OBJECTIVE: To analyze the clinical and molecular features of a child with carnitine palmitoyltransferase 1A (CPT1A) deficiency. METHODS: Clinical data of the child was collected. Blood acylcarnitine was determined with tandem mass spectrometry. DNA was extracted from the child and his parents. All exons and flanking regions of the CPT1A gene were analyzed by PCR and Sanger sequencing. RESULTS: Analysis showed that the patient carried compound heterozygous mutations c.1787T>C and c.2201T>C of the CPT1A gene, which derived his father and mother, respectively. Both mutations were verified as novel through the retrieval of dbSNP, HGMD and 1000 genome databases. Bioinformatic analysis suggested that the mutations can affect protein function. CONCLUSION: Acyl carnitine analysis has been the main method for the diagnosis of CPT1A deficiency. The c.1787T>C and c.2201T>C mutations of the CPT1A gene probably underlie the disease in this patient. Gene testing can provide important clues for genetic counseling and prenatal diagnosis.

[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.

[Relationship between expression of microRNA and inflammatory cytokines plasma level in pediatric patients with sepsis].

OBJECTIVE: Sepsis is the major cause of death in pediatric intensive care unit (PICU). The clinical manifestations of early sepsis is very similar to systemic inflammatory response syndrome (SIRS) caused by non-infectious reason. This study aimed to investigate the expression of miRNA and inflammatory cytokines in plasma in pediatric sepsis patients and its clinical significance. METHOD: Forty children with sepsis seen in Shenzhen children's hospital PICU from April 2012 to March 2013 were enrolled in this study, the median age was 0.75 (0.52, 1.90) years; 27 were males and 13 females, of whom 16 had severe sepsis. We selected 20 postsurgical patients with SIRS and 15 healthy children as a control group. The expression levels of plasma miR-21, miR-125b, miR-132, miR-146a, miR-155 and miR-223 were detected by real-time quantitative PCR (qRT-PCR). The predictive value of miRNA, PCT and CRP for sepsis were evaluated by Receiver operating characteristic curve (ROC). TNF-α and IL-10 levels in plasma detected by Cytometric Beads Array (CBA). Quantitative data of normal distribution was compared with ANOVA among the three groups and LSD-t test between two groups. To non-normal distribution of data, multiple comparisons among three groups were conducted by Kruskal-Wallis H test and differences between two groups were assessed by Mann-Whitney U test for post hoc analysis. RESULT: There were no significant differences between the age and gender of each group. Expression of miR-21, miR-125b, miR-132 and miR-155 in plasma had no significant difference in each group (all P > 0.05). MiR-146a and miR-223 levels in sepsis were upregulated compared with SIRS group and control group [(5.7 ± 3.5)×10(-5) vs. (2.4 ± 1.6)×10(-5) and (2.6 ± 1.2)×10(-5), (12.5 ± 7.7)×10(-4) vs. (8.3 ± 3.4)×10(-4) and (5.3 ± 2.2)×10(-4), all P < 0.01], expression levels of miR-223 in SIRS increased as compared to control group (P < 0.01). MiR-146a levels in severe sepsis were higher than those of the general sepsis [ (7.1 ± 3.3)×10(-5) vs. (4.6 ± 2.6)×10(-5), P < 0.01]. CRP and PCT levels are all higher in sepsis and SIRS groups than control group (all P < 0.01). The area under ROC curve (AUC) of miR-146a, miR-223, PCT and CRP to predict sepsis were 0.815 (95%CI: 0.708-0.922), 0.678(95%CI: 0.537-0.818), 0.706 (95%CI: 0.571-0.842) and 0.588 (95%CI: 0.427-0.748). Expression levels of IL-10 and IL-10/TNF-α in sepsis were upregulated compared with and SIRS group and the control group (all P < 0.01). There was a positive correlation between miR-146a, miR-223 and IL-10 and IL-10/TNF-α (r = 0.545, 0.305, 0.562, 0.373, all P < 0.01). CONCLUSION: The expression levels of miR-146a and miR-223 in plasma in pediatric patients with sepsis was significantly upregulated, and had a positive correlation with IL-10 and IL-10/TNF-α, which may be used as early diagnostic markers and can reflect the severity of condition to a certain degree.

[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.

[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.

A regulatory polymorphism in promoter region of TNFR1 gene is associated with Kawasaki disease in Chinese individuals.

Tumor necrosis factor (TNF) and the TNF receptor superfamily (TNF-TNFR) plays very important roles in the pathogenesis of Kawasaki disease (KD) by leukocyte recruitment, upregulation of matrix-degrading enzymes and proinflammatory cytokines. This study aims to investigate whether potential polymorphisms in TNF receptor superfamily member 1A gene (TNFR1) are associated with KD and its effects on transcriptions activity of TNFR1. Genetic variations of TNFR1 promoter and coding regions in 132 unrelated patients with KD and 212 age-matched healthy controls recruited from a population of Chinese individuals were screened by direct sequencing. Bioinformatics analysis and function assays were performed to investigate the association between genetic variations and KD, and its effects on transcription activity of TNFR1. Five polymorphisms, termed -609T/G, -581A/G, -421G/A, -383A/C, and +36A/G, were identified in the subjects, of which -421A/G was reported for the first time. In particular, bioinformatics analysis and function assay confirmed that -609T allele resulted in allele-specific strengthening of TNFR1 transcription and was significantly associated with KD (p = 2.951E-08, odds ratio = 2.42, 95% confidence interval = 1.76-3.13). Furthermore, the haplotype TAGAA showed a relatively higher frequency in patients with KD compared with healthy controls (p = 3.446E-07, odds ratio = 2.26, 95% confidence interval = 1.65-3.11). Therefore, our results suggested that regulatory polymorphism -609T/G and the haplotype TAGAA may be related to increased susceptibility to KD in Chinese individuals.

[Analysis of clinical features and gene mutations in two Chinese pedigrees with late-onset methylmalonic acidemia, cblC type].

OBJECTIVE: CblC is the most common type of methylmalonic acidemia with homocysteinemia. MMACHC is the coding gene. This study aimed at understanding clinical features and gene mutations in 2 Chinese pedigrees who had late-onset methylmalonic acidemia complicated with homocysteinemia. METHOD: The clinical data of 2 cases were analyzed. The MMACHC gene mutation was detected using polymerase chain reaction (PCR) and DNA sequencing. RESULT: The age of onset was 13 years and 12 years, respectively. They both presented with nervous system symptoms. The main clinical features were developmental retardation and degradation, including motion, speech and intelligence. One patient complained of anemia. The other patient was misdiagnosed as having a viral encephalitis. Both patients showed remarkable elevation of methylmalonic acid and homocysteine levels in urine. Both had received therapy with vitamin B(12). The symptoms were rapidly relieved. The follow-up till now showed apparent improvement in the 2 cases. Three mutations in the MMACHC gene were found in the two Chinese pedigrees. Both patients were compound heterozygotes of two mutant alleles: one patient had a G-to-A transition at nucleotide 482 (G482A) that caused an arginine-to-glutamine substitution at position 161 of the protein (R161Q), and a deletion of AAG at nucleotide 658_660 (658_660delAAG) which resulted in lysine deleting at position 220 of the protein (K220del); the other patient had a G482A and a G-to-A transition at nucleotide 609 (G609A) that caused a tryptophan-to-termination codon substitution at position 203 of the protein (W203X). Otherwise, the authors also detected parents of two families. Each had a heterozygote of one mutation. CONCLUSION: Late-onset methylmalonic acidemia patients had a variety of clinical manifestation, the first symptom was mainly abnormality of nervous system. One case was accompanied with hematological abnormalities. Two patients were vitamin B(12) responsive. In this study, the mutations were all detected on the fourth exon, the G482A mutation was probably associated with late-onset cases.