Evaluation of the clinical, biochemical, genotype and prognosis of mut-type methylmalonic acidemia in 365 Chinese cases.

BACKGROUND: Methylmalonic acidemia (MMA), which results from defects in methylmalonyl-CoA mutase (mut type) or its cofactor, is the most common inherited organic acid metabolic disease in China. This study aimed to investigate the phenotype and genotype of mut-type MMA in Chinese patients. METHODS: We recruited 365 patients with mut-type MMA; investigated their disease onset, newborn screening (NBS) status, biochemical metabolite levels, gene variations and prognosis; and explored the relationship between phenotype and genotype. RESULTS: There were 152 patients diagnosed by tandem mass spectrometry (MS/MS) expanded NBS, 209 patients diagnosed because of disease onset without NBS and 4 cases diagnosed because of sibling diagnosis. The median age of onset was 15 days old, with a variety of symptoms without specificity. Urinary levels of methylmalonic acid and methylcitric acid (MCA) decreased after treatment. Regarding the prognosis, among the 152 patients with NBS, 50.6% were healthy, 30.3% had neurocognitive impairment and/or movement disorders and 13.8% died. Among the 209 patients without NBS, 15.3% were healthy, 45.9% had neurocognitive impairment and/or movement disorders and 33.0% died. In total, 179 variants were detected in the MMUT gene, including 52 novel variations. c.729_730insTT, c.1106G>A, c.323G>A, c.914T>C and c.1663G>A were the five most frequent variations. The c.1663G>A variation led to a milder phenotype and better prognosis. CONCLUSION: There is a wide spectrum of variations in the MMUT gene with several common variations. Although the overall prognosis of mut-type MMA was poor, participation in MS/MS expanded NBS, vitamin B12 responsive and late onset are favourable factors for the prognosis.

Diagnosis and follow-up of glycogen storage disease (GSD) type VI from the largest GSD center in China.

Glycogen storage disease (GSD) Type VI is a glycogenolysis disorder caused by variants of PYGL. Knowledge about this disease is limited because only approximately 50 cases have been reported. We investigated the clinical profiles, molecular diagnosis, and treatment outcomes in patients with GSD VI from 2000 to 2021. The main initial clinical features of this cohort include hepatomegaly, short stature, elevated liver transaminases, hypertriglyceridemia, fasting hypoglycemia, and hyperuricemia. After uncooked cornstarch treatment, the stature and biochemical parameters improved significantly (p < 0.05). However, hyperuricemia recurred in most patients during adolescence. Among the 56 GSD VI patients, 54 biallelic variants and two single allelic variants of PYGL were identified, of which 43 were novel. There were two hotspot variants, c.1621-258_2178-23del and c.2467C>T p.(Gln823*), mainly in patients from Southwest and South China. c.1621-258_2178-23del is a 3.6 kb deletion that results in an out-of-frame deletion r.1621_2177del and an in-frame deletion r.1621_2265del. Our data show for the first time that long-term monitoring of uric acid is recommended for older GSD VI patients. This study also broadens the variant spectrum of PYGL and indicates that there are two hot-spot variants in China.

Clinical characteristics and genotype analysis of five infants with cblX type of methylmalonic acidemia.

OBJECTIVE: To investigate the clinical and genetic characteristics of infants with cobalamin (cbl) X type of methylmalonic acidemia (MMA). METHODS: The clinical data of 5 infants with cblX type of MMA diagnosed in Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine and Shanghai Children's Hospital from the year 2016 to 2020 were collected. The levels of blood acylcarnitines were detected by tandem mass spectrometry, the levels of urinary organic acids were detected by gas-chromatography mass spectrometry, the pathogenic genes were detected by whole exon gene sequencing, and the effect of new pathogenic mutations on three-dimensional protein structure was predicted by bioinformatics analysis. RESULTS: Five infants with cblX type were diagnosed, including 4 males and 1 female, and the onset age was 0-6 months. The main clinical manifestations of 4 males were intractable epilepsy, mental and motor retardation, metabolic abnormalities presented mild increase of blood homocysteine level. Among them, 3 cases were accompanied by slight increase of urinary methylmalonic acid, and 1 case was accompanied by increase of blood propionylcarnitine (C3) and C3/acetylcarnitine (C2). Gene detection found that 2 cases carried a same hemizygous mutation c.344C>T (p.A115V) of HCFC1 gene, which was the most reported mutation, and the other 2 cases carried novel pathogenic mutations, c.92G>A (p.R31Q) and c.166G>C (p.V56L). These 3 gene mutations located in the Kelch domain of HCFC1 protein. One female infant carried a benign mutation of c.3731G>T (p.R1244L). Her clinical symptoms were mild, and only the urinary methylmalonic acid was slightly increased. CONCLUSIONS: The clinical manifestations of children with cblX type of MMA are intractable epilepsy, mental and motor retardation, and other serious neurological symptoms. Their metabolic abnormalities present the increase of blood homocysteine with methylmalonic acid (urinary methylmalonic acid or/and blood C3, C3/C2). The clinical and biochemical phenotypes are separated, so the diagnosis should be in combination with the results of gene testing.

Diagnostic yield of additional exome sequencing after the detection of long continuous stretches of homozygosity (LCSH) in SNP arrays.

Long continuous stretches of homozygosity (LCSH) are associated with risk of recessive disorders. Though LCSH can be detected by SNP microarrays, additional testing is necessary to clarify the clinical significance. This study is to assess the yield of additional exome sequencing (ES) after LCSH detection and inform the likelihood of eventual diagnosis. In 2226 patients referred to SNP microarrays, 35 patients met the criteria of indicative LCSH. These patients were recruited and went through additional ES. The diagnostic yield was analyzed, and the LCSH pattern was compared between eventually diagnosed cases and those undiagnosed. The results showed additional ES attained a diagnostic yield of 31.4% (11/35), but only one-third of the yield (11.4%, 4/35) was relevant to LCSH. In contrast, two-thirds of the diagnostic variants (20%, 7/35) were de novo or dominantly inherited, irrelevant to the original LCSH finding. No particular LCSH pattern, including the chromosomal coverage or LCSH size, was found to associate with the diagnostic outcome. We concluded that additional ES after LCSH detection could reveal diagnostic variants, but it is strongly recommended to consider all possible inheritance mode, as the diagnostic variants may be irrelevant to the original LCSH finding.

High Detection Rate of Copy Number Variations Using Capture Sequencing Data: A Retrospective Study.

BACKGROUND: Capture sequencing (CS) is widely applied to detect small genetic variations such as single nucleotide variants or indels. Algorithms based on depth comparison are becoming available for detecting copy number variation (CNV) from CS data. However, a systematic evaluation with a large sample size has not been conducted to evaluate the efficacy of CS-based CNV detection in clinical diagnosis. METHODS: We retrospectively studied 3010 samples referred to our diagnostic laboratory for CS testing. We used 68 chromosomal microarray analysis-positive samples (true set [TS]) and 1520 reference samples to build a robust CS-CNV pipeline. The pipeline was used to detect candidate clinically relevant CNVs in 1422 undiagnosed samples (undiagnosed set [UDS]). The candidate CNVs were confirmed by an alternative method. RESULTS: The CS-CNV pipeline detected 78 of 79 clinically relevant CNVs in TS samples, with analytical sensitivity of 98.7% and positive predictive value of 49.4%. Candidate clinically relevant CNVs were identified in 106 UDS samples. CNVs were confirmed in 96 patients (90.6%). The diagnostic yield was 6.8%. The molecular etiology includes aneuploid (n = 7), microdeletion/microduplication syndrome (n = 40), and Mendelian disorders (n = 49). CONCLUSIONS: These findings demonstrate the high yield of CS-based CNV. With further improvement of our CS-CNV pipeline, the method may have clinical utility for simultaneous evaluation of CNVs and small variations in samples referred for pre- or postnatal analysis.

Identification of RUNX2 variants associated with cleidocranial dysplasia.

BACKGROUND: Cleidocranial dysplasia (CCD) is a rare autosomal dominant disorder mainly characterized by hypoplastic or absent clavicles, delayed closure of the fontanelles, multiple dental abnormalities, and short stature. Runt-related transcription factor 2 (RUNX2) gene variants can cause CCD, but are not identified in all CCD patients. METHODS: In this study, we detected genetic variants in seven unrelated children with CCD by targeted high-throughput DNA sequencing or Sanger sequencing. RESULTS: All patients carried a RUNX2 variant, totally including three novel pathogenic variants (c.722_725delTGTT, p.Leu241Serfs*8; c.231_232delTG, Ala78Glyfs*82; c.909C > G, p.Tyr303*), three reported pathogenic variants (c.577C > T, p.Arg193*; c.574G > A, p.Gly192Arg; c.673 C > T, p.Arg225Trp), one likely pathogenic variant (c.668G > T, p.Gly223Val). The analysis of the variant source showed that all variants were de novo except the two variants (c.909C > G, p.Tyr303*; c.668G > T, p.Gly223Val) inherited from the patient's father and mother with CCD respectively. Further bioinformatics analysis indicated that these variants could influence the structure of RUNX2 protein by changing the number of H-bonds or amino acids. The experimental result showed that the Gly223Val mutation made RUNX2 protein unable to quantitatively accumulate in the nucleus. CONCLUSIONS: The present study expands the pathogenic variant spectrum of RUNX2 gene, which will contribute to the diagnosis of CCD and better genetic counseling in the future.

Genetic Evaluation of 114 Chinese Short Stature Children in the Next Generation Era: a Single Center Study.

BACKGROUND/AIMS: The genetics of human height is a frequently studied and complex issue. However, there is limited genetic research of short stature. To uncover the subgroup of patients to have higher yield and to propose a simplified diagnostic algorithm in the next generation era. METHODS: This study included 114 Chinese children with height SDS ≤ -2.5 and unknown etiology from 2014 to 2015. Target/whole exome sequencing (referred as NGS) and chromosomal microarray analysis (CMA) were performed on the enrolled patients sequentially to identify potential genetic etiologies. The samples solved by NGS and CMA were retrospectively studied to evaluate the clinical pathway of the patients following a standard diagnostic algorithm. RESULTS: In total, a potential genetic etiology was identified in 41 (36%) patients: 38 by NGS (33.3%), two by CMA (1.8%), and an additional one by both (0.9%). There were 46 different variants in 29 genes and 2 pathogenic CNVs identified. The diagnostic yield was significantly higher in patients with facial dysmorphism or skeletal abnormalities than those without the corresponding phenotype (P=0.006 and P=0.009, respectively, Pearson's χ2 test). Retrospectively study the cohort indicate 83.3% patients eventually would be evaluated by NGS/CMA. CONCLUSION: This study confirms the utility of high-throughput molecular detection techniques for the etiological diagnosis of undiagnosed short stature and suggests that NGS could be used as a primary diagnostic strategy. Patients with facial dysmorphism and/or skeletal abnormalities are more likely to have a known genetic etiology. Moving NGS forward would simplified the diagnostic algorithm.

Marked yield of re-evaluating phenotype and exome/target sequencing data in 33 individuals with intellectual disabilities.

The diagnosis of intellectual disability/developmental delay (ID/DD) benefits from the clinical application of target/exome sequencing. The yield in Mendelian diseases varies from 25% to 68%. The aim of the present study was to identify the genetic causes of 33 ID/DD patients using target/exome sequencing. Recent studies have demonstrated that reanalyzing undiagnosed exomes could yield additional diagnosis. Therefore, in addition to the normal data analysis, in this study, re-evaluation was performed prior to manuscript preparation after updating OMIM annotations, calling copy number variations (CNVs) and reviewing the current literature. Molecular diagnosis was obtained for 19/33 patients in the first round of analysis. Notably, five patients were diagnosed during the re-evaluation of the geno/phenotypic data. This study confirmed the utility of exome sequencing in the diagnosis of ID/DD. Furthermore, re-evaluation leads to a 15% improvement in diagnostic yield. Thus, to maximize the diagnostic yield of next-generation sequencing (NGS), periodical re-evaluation of the geno/phenotypic data of undiagnosed individuals is recommended by updating the OMIM annotation, applying new algorithms, reviewing the literature, sharing pheno/genotypic data, and re-contacting patients.

Biochemical, molecular and outcome analysis of eight chinese asymptomatic individuals with methyl malonic acidemia detected through newborn screening.

Methyl malonic academia (MMA) is characterized by abnormal accumulation of methyl malonic acid in body fluids. Patients usually have a variety of clinical symptoms including recurrent vomiting, metabolic acidosis, developmental delay, seizure, or death. However, a few cases where the patients have no symptom are also reported. Here, we conducted clinical, biochemical, and molecular analysis of eight Chinese patients identified through newborn screening between 2003 and 2013. All the patients had significantly higher blood propionylcarnitine (C3) concentrations, ratio of propionylcarnitine/acetylcarnitine (C3/C2); and their urine methyl malonic acid and methylcitric acid (MCA) excretions were remarkably higher than normal at diagnosis and during follow-ups. In addition, five different known mutations were identified in seven of the eight patients in either MUT or MMACHC. All these mutations were expected to produce defective proteins that would result in decreased or even total loss of methyl malonyl-CoA mutase activity. However, normal outcomes were found in all patients in physical growth, intellectual performance and cerebral MRI analysis at diagnosis (range, 14-53 days) and during follow-ups (range, 1.8-10 years). Our study is the first report of Chinese MMA patients with increased secretion of methyl malonic acid and molecular defects in MUT or MMACHC yet remain asymptomatic.

[Molecular and prenatal diagnosis of a family with Fanconi anemia by next generation sequencing].

OBJECTIVE: To provide prenatal diagnosis for a pregnant woman who had given birth to a child with Fanconi anemia with combined next-generation sequencing (NGS) and Sanger sequencing. METHODS: For the affected child, potential mutations of the FANCA gene were analyzed with NGS. Suspected mutation was verified with Sanger sequencing. For prenatal diagnosis, genomic DNA was extracted from cultured fetal amniotic fluid cells and subjected to analysis of the same mutations. RESULTS: A low-frequency frameshifting mutation c.989_995del7 (p.H330LfsX2, inherited from his father) and a truncating mutation c.3971C>T (p.P1324L, inherited from his mother) have been identified in the affected child and considered to be pathogenic. The two mutations were subsequently verified by Sanger sequencing. Upon prenatal diagnosis, the fetus was found to carry two mutations. CONCLUSION: The combined next-generation sequencing and Sanger sequencing can reduce the time for diagnosis and identify subtypes of Fanconi anemia and the mutational sites, which has enabled reliable prenatal diagnosis of this disease.

[Clinical characteristics and analysis of mass spectrometric data in patients with ornithine transcarbamylase deficiency].

OBJECTIVE: To explore the clinical manifestations and biochemical characteristics of patients with ornithine transcarbamylase deficiency (OTCD) so as to increase the clinician awareness for this disease. METHODS: The clinical manifestations, blood ammonia levels, citrulline levels, urinary orotic acid and uracil levels were analyzed for 40 patients with OTCD from 2005 to 2013. And comparisons were made with 25 healthy children. RESULTS: Among them, the median age of onset was 1.4 years (3 days-29 years). The major clinical manifestations were feeding difficulties, persistent vomiting, convulsions, unconsciousness and hyperammonemia, etc. The blood levels of citrulline in these patients were significantly lower than those of the control group (6.35 (1.84-21.11) vs 13.65 (10.23-24.52) µmol/L, P < 0.05) . The urinary levels of orotic acid and uracil in these patients were significantly higher than those in the control group (167.77 (1.21-1 650.45) vs 0.25 (0-2.32) mmol/molCr, 52.67 (3.50-338.64) vs 0.69 (0-2.87) mmol/molCr, P < 0.05) . CONCLUSION: For patients with hyperammonemia, the decreased levels of citrulline in blood tested by tandem mass spectrometry and increased orotic acid and uracil in urine on gas chromatography-mass spectrometry may aid the diagnosis OTCD.

Clinical, genetic profile and therapy evaluation of 11 Chinese pediatric patients with Fanconi-Bickel syndrome.

BACKGROUND: Fanconi-Bickel syndrome (FBS) is a rare autosomal recessive disorder characterized by impaired glucose and galactose utilization as well as proximal renal tubular dysfunction. METHODS: Clinical, biochemical, genetic, treatment, and follow-up data for 11 pediatric patients with FBS were retrospectively analysed. RESULTS: Hepatomegaly (10/11), short stature (10/11) and hypophosphataemic rickets (7/11) were the most common initial symptoms. At diagnosis, all patients had decreased fasting blood glucose (FBG), plasma bicarbonate (HCO3-) and serum phosphorus, as well as elevated liver transaminases, alkaline phosphatase (AKP) and proximal renal tubular dysfunction. Two infant patients were misdiagnosed with transient neonatal diabetes mellitus. After therapy with uncooked cornstarch and conventional rickets treatment, remission of hepatomegaly was observed in all patients, with significant improvements in pre-prandial blood glucose, liver transaminases, triglyceride, plasma HCO3- and AKP (p < 0.05). At the last follow-up, 5/7 patients with elevated AKP had nephrocalcinosis. The mean height standard deviation score (Ht SDS) of eight patients with regular treatment increased from - 4.1 to -3.5 (p = 0.02). Recombinant human growth hormone (rhGH) was administered to 4/9 patients, but their Ht SDS did not improve significantly (p = 0.13). Fourteen variants of the SLC2A2 gene were identified, with six being novel, among which one was recurrent: c.1217T > G (p.L406R) (allele frequency: 4/22, 18%). Patients with biallelic missense variants showed milder metabolic acidosis than those with null variants. Two of five patients from nonconsanguineous families with rare homozygous variations showed 5.3 Mb and 36.6 Mb of homozygosity surrounding the variants, respectively; a region of homozygosity (ROH) involving the entire chromosome 3 covering the SLC2A2 gene, suggesting uniparental disomy 3, was detected in one patient. CONCLUSIONS: Early diagnosis of FBS is difficult due to the heterogeneity of initial symptoms. Although short stature is a major issue of treatment for FBS, rhGH is not recommended in FBS patients who have normal GH stimulation tests. Patients with biallelic null variants may require alkali supplementation since urine bicarbonate loss is genetically related. ROH is a mechanism for rare homozygous variants of FBS in nonconsanguineous families.

A Novel Mutation in Melanocortin Receptor 2 and a Reported Mutation in Melanocortin Receptor 2 Accessory Protein: Three Chinese Cases with Familial Glucocorticoid Deficiency.

Background: Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disease characterized by glucocorticoid deficiency without mineralocorticoid deficiency. We report 3 Chinese patients with MRAP or MC2R mutations. Case Reports: Patient 1 presented with hyperpigmentation. Endocrine investigations revealed low serum cortisol levels and elevated adrenocorticotropic hormone (ACTH) levels. Furthermore, low serum sodium was evident. She was diagnosed with FGD type 2 due to a homozygous mutation in MRAP (c.106+1delG), revealed through exome sequencing (ES). After 2-year treatment with hydrocortisone, skin hyperpigmentation was improved. Patient 2 initially presented with hyponatremia. Low cortisol levels and high levels of ACTH were subsequently detected; he was subjected to a hydrocortisone treatment during which he experienced repeated hypoglycemic attacks and pigmentation. ES revealed the same mutation as in patient 1 in MRAP (c.106+1delG), thus he was diagnosed with FGD type 2. After 6 years of age, his symptoms remarkably improved, and there was no episode of hypoglycemia. Patient 3 mainly presented with hyperpigmentation, hypoglycemic attack, and tall stature. Laboratory findings were normal except for low serum cortisol levels and high ACTH levels. She was diagnosed with FGD type 1 as ES revealed a novel homozygous mutation in MC2R (c.712C>A, p.His238Tyr). After nearly 2 years of hydrocortisone replacement therapy, the excessive growth was reduced to near normal, and the skin color returned to normal. Conclusions: Three patients were diagnosed with FGD (one with FGD type 1 and two with FGD type 2). They all presented with hyperpigmentation and hypoglycemia; however, compared with patient 1, the clinical manifestations of patient 2 were more complicated. Patient 3 had later onset and taller stature than patients 1 and 2. A novel mutation in patient 3 expands the mutation spectrum of MC2R.

Deep Intronic PAH Variants Explain Missing Heritability in Hyperphenylalaninemia.

Phenylalanine hydroxylase (PAH) deficiency or phenylketonuria (PKU) is the most common cause of hyperphenylalaninemia (HPA), and approximately 5% of patients remain genetically unsolved. Identifying deep intronic PAH variants may help improve their molecular diagnostic rate. Next-generation sequencing was utilized to detect the whole PAH gene in 96 patients with genetically unsolved HPA from 2013 to 2022. The effects of deep intronic variants on pre-mRNA splicing were investigated by minigene-based assay. The allelic phenotype values of recurrent deep intronic variants were calculated. Twelve deep intronic PAH variants, located in intron 5 (c.509+434C>T), intron 6 (c.706+288T>G, c.706+519T>C, c.706+531T>C, c.706+535G>T, c.706+600A>C, c.706+603T>G, and c.706+608A>C), intron 10 (c.1065+241C>A and c.1065+258C>A), and intron 11 (c.1199+502A>T and c.1199+745T>A) were identified in 80.2% (77/96) patients. Ten of the 12 variants were novel, and they all generated pseudoexons in mRNA, leading to frameshift or lengthened proteins. The most prevalent deep intronic variant was c.1199+502A>T, followed by c.1065+241C>A, c.1065+258C>A, and c.706+531T>C. The metabolic phenotypes of the four variants were assigned as classic PKU, mild HPA, mild HPA, and mild PKU, respectively. The results suggest that deep intronic PAH variants improved the diagnostic rate from 95.3% to 99.3% in the overall patients with HPA. Our data demonstrate the importance of assessing noncoding variants in genetic diseases. Pseudoexon inclusion caused by deep intronic variants may represent a recurrent mechanism.

Clinical and genetic characteristics of 42 Chinese paediatric patients with X-linked adrenal hypoplasia congenita.

BACKGROUND: X-linked adrenal hypoplasia congenita (AHC) is a rare disorder characterized by primary adrenal insufficiency (PAI) and hypogonadotropic hypogonadism (HH), with limited clinical and genetic characterization. METHODS: The clinical, biochemical, genetic, therapeutic, and follow-up data of 42 patients diagnosed with X-linked AHC were retrospectively analysed. RESULTS: Hyperpigmentation (38/42, 90%), vomiting/diarrhoea (20/42, 48%), failure to thrive (13/42, 31%), and convulsions (7/42, 17%) were the most common symptoms of X-linked AHC at onset. Increased adrenocorticotropic hormone (ACTH) (42/42, 100%) and decreased cortisol (37/42, 88%) were the most common laboratory findings, followed by hyponatremia (32/42, 76%) and hyperkalaemia (29/42, 69%). Thirty-one patients presented with PAI within the first year of life, and 11 presented after three years of age. Three of the thirteen patients over the age of 14 exhibited spontaneous pubertal development, and ten of them experienced delayed puberty due to HH. Six patients receiving human chorionic gonadotropin (hCG) therapy exhibited a slight increase in testicular size and had rising testosterone levels (both P < 0.05). The testicular volumes of the three patients with pulsatile gonadotropin-releasing hormone (GnRH) therapy were larger than those of the six patients undergoing hCG therapy (P < 0.05), and they also exhibited some growth in terms of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone. Of the 42 patients, three had an Xp21 deletion, and 39 had an isolated DAX1 defect. Most patients (9/10) with entire DAX1 deletion accounting for 23.8% (10/42) of the total variants had early onset age of less than one year. CONCLUSIONS: This study details the clinical features and genetic spectra of X-linked AHC. Patients with X-linked AHC show a bimodal distribution of the age of onset, with approximately 70% presenting within the first year of life. Pulsatile GnRH may be recommended for HH when hCG therapy is not satisfactory, although it is difficult to achieve normal testicular volume. The combination of clinical features and molecular tests provides information for an accurate diagnosis.

Variable phenotypes and outcomes associated with the MMACHC c.482G > A mutation: follow-up in a large CblC disease cohort.

BACKGROUND: The aim of this study was to characterize the variable phenotypes and outcomes associated with the methylmalonic aciduria and homocystinuria type C protein gene (MMACHC) c.482G > A mutation in 195 Chinese cases with CblC disease. METHODS: We carried out a national, retrospective multicenter study of 195 Chinese patients with CblC disease attributable to the MMACHC c.482G > A variant either in a homozygous or compound heterozygous state. The control group consisted of 200 patients diagnosed with CblC disease who did not possess the c.482G > A mutation. Clinical features, including disease onset, symptoms, biochemical metabolites, gene mutation, and follow-up outcomes were reviewed and analyzed in detail. The median follow-up period spanned 3 years and 8 months, with a range of 1 year and 2 months to 12 years and 10 months. RESULTS: Among 195 patients carrying the c.482G > A variant, 125 (64.1%) cases were diagnosed by newborn screening (NBS), 60 (30.8%) cases were detected due to disease onset, and 10 (5.1%) cases were identified from sibling diagnoses. One hundred and seventeen (93.6%) individuals who were diagnosed by NBS, and nine patients who came from sibling diagnoses remained asymptomatic in this study. From 69 symptomatic patients of the c.482G > A group, more patients presented with later onset, and the top six common clinical symptoms at disease onset were developmental delay (59.4%), lower limb weakness and poor exercise tolerance (50.7%), cognitive decline (37.7%), gait instability and abnormal posture (36.2%), seizures (26.1%), and psychiatric and behavioral disturbances (24.6%). In the 159 symptomatic patients lacking c.482G > A variants, the most frequently observed clinical manifestations at disease onset included developmental delay (81.8%), lethargy and feeding difficulty (62.9%), lower limb weakness and poor exercise tolerance (54.7%), prolonged neonatal jaundice (51.6%), vomiting (47.2%), and seizures (32.7%). Before treatment, the levels of blood propionylcarnitine, propionylcarnitine/acetylcarnitine ratio, and homocysteine in the c.482G > A group were significantly lower (P < 0.05) than those in the non-c.482G > A group, while the concentration of urinary methylmalonic acid was slightly lower (P > 0.05). The degree of decline in the above metabolites after treatment in different groups significantly differed in both plasma total homocysteine values and urinary methylmalonic acid levels (P < 0.05). In patients carrying the c.482G > A variant compared with the non-c.428G > A group, there were markedly lower rates of mortality (0.5% vs. 2.0%) and developmental delay (20.5% vs. 65.5%). When compared with individuals diagnosed due to disease onset, those identified through NBS in either group exhibited a reduced proportion of disease onset (6.7% vs. 100% in the c.482G > A group, 54.4% vs. 100% in the non-c.482G > A group), lower mortality (0.0% vs. 1.7% in the c.482G > A group, 0.0% vs. 3.6% in the non-c.482G > A group), and had a higher percentage of patients exhibiting normal psychomotor and language development (99.3% vs. 33.3% in the c.482G > A group, 58.9% vs. 10.9% in the non-c.482G > A group). CONCLUSIONS: The c.482G > A variant in MMACHC is associated with late-onset and milder phenotypes of CblC disease. Patients with this mutation tend to have a relatively better response to hydroxocobalamin, better metabolic control, and more favorable neurological outcomes. NBS and other appropriate pre-symptomatic treatments seem to be helpful in early diagnosis, resulting in favorable clinical outcomes. Video Abstract (MP4 136794 kb).

Analysis of gene mutations in Chinese patients with maple syrup urine disease.

OBJECTIVE: Maple syrup urine disease (MSUD) is predominantly caused by mutations in the BCKDHA, BCKDHB and DBT genes, which encode for the E1α, E1ß and E2 subunits of the branched-chain α-keto acid dehydrogenase complex, respectively. The aim of this study was to screen DNA samples from 16 Chinese MSUD patients and assess a potential correlation between genotype and phenotype. METHODS: BCKDHA, BCKDHB and DBT genes were analyzed by polymerase chain reaction (PCR) and direct sequencing. Segments bearing novel mutations were identified by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. RESULTS: Within the variant alleles, 28 mutations (28/32, 87.5%), were detected in 15 patients, while one patient displayed no mutations. Mutations were comprised of 20 different: 6 BCKDHA gene mutations in 4 cases, 10 BCKDHB gene mutations in 8 cases and 4 DBT gene mutations in 3 cases. From these, 14 were novel, which included 3 mutations in the BCKDHA gene, 7 in the BCKDHB gene and 4 in the DBT gene. Only two patients with mutations in the BCKDHB and DBT genes were thiamine-responsive and presented a better clinical outcome. CONCLUSION: We identified 20 different mutations within the BCKDHA, BCKDHB and DBT genes among 16 Chinese MSUD patients, including 14 novel mutations. The majority were non-responsive to thiamine, associating with a worse clinical outcome. Our data provide the basis for further genotype-phenotype correlation studies in these patients, which will be beneficial for early diagnosis and in directing the approach to clinical intervention.

[Analysis of clinical data and genetic mutations in three Chinese patients with tyrosinemia type I].

OBJECTIVE: To analyze clinical data and gene mutations in 3 Chinese patients with tyrosinemia type I, and to explore the correlation between genotypes and phenotypes. METHODS: Three patients suspected with tyrosinemia I were tested by tandem mass spectrometry for the level of tyrosine, phenylalanine and succinylacetone in the blood, and by gas chromatography-mass spectrometry to determine the level of succinylacetone and organic acid in their urine. With the diagnosis established, the FAH gene was analyzed with polymerase chain reaction (PCR) and direct sequencing. RESULTS: Two patients had acute onset of the disease, while another had subacute onset of the disease, with features including hepatomegaly and remarkably increased tyrosine and succinylacetone in the blood. Five mutations were detected in the FAH gene, which included c.455G>A (W152X), c.520C>T (R174X), c.974_976delCGAinsGC, c.1027 G>A (G343R) and c.1100 G>A (W367X), among which c.455G>A (W152X), c.974_976delCGAinsGC and c.1100 G>A (W367X) were not reported previously. CONCLUSION: Tyrosinemia type I may be effectively diagnosed with the level of tyrosine and succinylacetone by tandem mass spectrometry and succinylacetone in the urine by gas chromatography mass spectrometry. Detection of underlying mutations mutations will be helpful for genetic counseling and further research.

Prenatal Diagnosis of Isovaleric Acidemia From Amniotic Fluid Using Genetic and Biochemical Approaches.

Background: Isovaleric acidemia (IVA) is an inborn error of leucine metabolism and different approaches have been applied to its prenatal diagnosis. However, systemic application of a biochemical strategy is rare. To evaluate its reliability and validity, we conducted a retrospective study of our experience with metabolite measurement together with genetic analysis in IVA prenatal diagnosis at a single center. Methods: A total of eight pregnancies whose probands were diagnosed as IVA were referred to our center for prenatal diagnosis. Prenatal data of genetic analysis and metabolite measurement using tandem mass spectrometry (MS/MS) and gas chromatography/mass spectrometry (GC/MS) in amniotic fluid (AF) samples were retrospectively reviewed. Results: Genetic and biochemical results were both available in these eight at-risk fetuses. Among them, two fetuses had higher levels of isovalerylcarnitine (C5) and C5/acetylcarnitine (C2) in AF compared with normal reference range and, thus, were determined to be affected, both of whom were found to carry compound heterogeneous mutations according to genetic analysis. The remaining six fetuses were determined to be unaffected based on a normal AF metabolite profile, except one showed slightly elevated C5 and they were found to be carriers according to genetic analysis. However, the level of isovalerylglycine (IVG) could not be detected at all in both groups. Conclusion: The biochemical analysis, as a quick and convenient method, could be an additional reliable option for the prenatal diagnosis of IVA, especially in families with inconclusive genetic results, and can achieve a more precise diagnosis in conjunction with mutation analysis.

The Follow-Up of Chinese Patients in cblC Type Methylmalonic Acidemia Identified Through Expanded Newborn Screening.

Objective: The cblC type of combined methylmalonic acidemia and homocystinuria, an inherited disorder with variable phenotypes, is included in newborn screening (NBS) programs at multiple newborn screening centers in China. The present study aimed to investigate the long-term clinical benefits of screening individual. Methods: A national, retrospective multi-center study of infants with confirmed cblC defect identified by NBS between 2004 and 2020 was conducted. We collected a large cohort of 538 patients and investigated their clinical data in detail, including disease onset, biochemical metabolites, and gene variation, and explored different factors on the prognosis. Results: The long-term outcomes of all patients were evaluated, representing 44.6% for poor outcomes. In our comparison of patients with already occurring clinical signs before treatment to asymptomatic ones, the incidence of intellectual impairment, movement disorders, ocular complications, hydrocephalus, and death were significantly different (p < 0.01). The presence of disease onset [Odd ratio (OR) 12.39, 95% CI 5.15-29.81; p = 0.000], variants of c.609G>A (OR 2.55, 95% CI 1.49-4.35; p = 0.001), and c.567dupT (OR 2.28, 95% CI 1.03-5.05; p = 0.042) were independently associated with poor outcomes, especially for neurodevelopmental deterioration. Conclusion: NBS, avoiding major disease-related events and allowing an earlier treatment initiation, appeared to have protective effects on the prognosis of infants with cblC defect.