Zhuwen Gong

Identification of a distinct mutation spectrum in the SMPD1 gene of Chinese patients with acid sphingomyelinase-deficient Niemann-Pick disease

BackgroundClinical observations and molecular analysis of the SMPD1 gene in Chinese patients with acid sphingomyelinase deficiency Niemann-Pick disease (NPD) are scarce.MethodsA cohort of 27 Chinese patients diagnosed with acid sphingomyelinase deficiency, within the past five years, were collected and investigated for genotype, phenotype, and their correlations.ResultsThe majority of our patients (25/27) were under 18 years of age. From the cohort group, eight (30%) fulfilled characters of type A. Four other patients experienced neurologic involvement after two years of age, these were classified as intermediate type. The remaining fifteen presented without clear neurologic involvement and were regarded as type B. One patient, from the type B group, presented with the unusual symptom of a secondary amenorrhea. Three patients, one from the type B group and two from the intermediate group, presented with pronounced proteinuria, in the late stages of the disease, indicating possible kidney involvement in NPD. Twenty-four SMPD1 gene mutations had been identified; eighteen of these are novel ones. These included four exonic small deletions/duplications (c.4delC, c.147_150del4, c.842-849dup8, c.1307-1312dup6), one termination mutation (p.Glu248X), and thirteen exonic point mutations (p.Gly336Ser, p.Trp342Cys, p.Leu382Phe, p.Pro429Leu, p.Pro430Ser, p.Trp437Arg, p.Thr451Pro, p.His461Pro, p.Ala484Val, p.Ser486Arg, p.Tyr500His, p.Pro533Leu, p.Val559Leu). Notably, eight mutations had more than one occurrence with c.4delC and p.Glu248X accounting for ~30% of all alleles. Correlation analysis of genotype and phenotype indicated eight mutations, c.842-849dup8, p.Glu248X, p.Arg230Cys, p.Trp437Arg, p.His461Pro, p.Ala484Val p.Ser486Arg, and p.Pro533Leu,to be severe mutations. Five mutations, c.4delC, p.Leu382Phe, p.Pro429Leu, p.Pro430Ser and p.Val559Leu were projected to be mild mutations. Interestingly, three intermediate individuals carried combinations of a mild mutation, c.4delC, on one allele and a severe mutation on the other allele.ConclusionsThe Chinese population may have a comparably high incidence of sphingomyelinase-deficient Niemann-Pick disease type A. This study has identified some novel genotype and phenotype correlations in this rare and devastating disorder.

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, E1β 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.

Patients with progressive pseudorheumatoid dysplasia: from clinical diagnosis to molecular studies.

Progressive pseudorheumatoid dysplasia (PPD) is a rare inherited autosomal recessive disease for which no prevalent data have been reported in China. We aimed to identify PPD based on clinical manifestations and imaging analysis of the bony skeleton and then to investigate gene mutations of Wnt1-inducible signaling pathway protein 3 (WISP3) in Chinese patients with PPD. Seven patients (aged 9-49 years) from six unrelated Chinese families all presented with a waddling gait, progressive swelling and restricted joint movements, and all were diagnosed as having PPD according to clinical signs and symptoms, as well as radiographic imaging. The radiographic imaging revealed no erosive arthropathy, but showed platyspondyly, irregular or wedged/ovoid anterior end-plates of the vertebral bodies, coxa vara and widened epiphyses or metaphyses including the femoral head and the metacarpophalangeal and interphalangeal joints. Normal laboratory values were found for the erythrocyte sedimentation rate, C-reactive protein and rheumatoid factors in all patients. Molecular studies revealed that five patients carried c.624_625insA/c.729_735delGAGAAAA, c.624_625insA/c.866_867insA, c.866_867 insA/c.866_867insA, Q46X/C114W and C223G/C114W mutations, respectively. In conclusion, our findings suggest that in order to avoid misdiagnosis, physicians should carefully examine the entire skeleton, including the spine, in addition to the skeletal extremities. Mutation analysis of the WISP3 gene is useful for confirming the clinical and radiographic diagnosis of PPD.

21-hydroxylase deficiency-induced congenital adrenal hyperplasia in 230 Chinese patients: Genotype-phenotype correlation and identification of nine novel mutations.

Steroid 21-hydroxylase deficiency (21-OHD) caused by the CYP21A2 gene mutations accounts for more than 90% of congenital adrenal hyperplasia (CAH) cases. In this study, molecular defects of 230 patients with 21-OHD were investigated. Point mutations of CYP21A2 gene were analyzed by Sanger sequencing, and large gene deletions were detected by multiplex ligation-dependent probe amplification (MLPA). Nine micro-conversions and 18 spontaneous mutations accounted for 74.6% of alleles, while large gene deletions and large gene conversions accounted for 25.4% of alleles. The most frequent micro-conversion was c.292-13A/C>G (I2G) (35%), followed by p.I173N (14.3%), p.R357W (5.9%) and p.Q319* (4.6%). Nine novel mutations were identified in these patients, which were predicted to hamper the 21-hydroxylase protein function in varying degrees. Genotype and phenotype correlated well in 89.6% of our patients, but disparity in phenotypic appearance also appeared in a small portion of the patients. 16.1% of the patients carried homozygous genotypes while 83.9% of patients carried compound heterozygous mutations. We concluded that the frequency of CYP21A2 mutations in our study was slightly different from those reported for other ethnic groups. Micro-conversions were the main category of the mutation spectrum, while large deletions and large gene conversions could also cause 21-OHD. A large portion of different types of the compound heterozygous genotypes may partially contribute to the discordance in genotype-phenotype comparison. This study expanded the CYP21A2 mutation spectrum of Chinese patients and could be helpful in prenatal diagnosis and genetic counseling for 21-OHD patients.

Maternal origin of a de novo microdeletion spanning the ERCC6 gene in a classic form of the Cockayne syndrome

The Cockayne syndrome is a rare autosomal recessive disease characterized by a general developmental delay, the unique face, and abnormal skin sensitivity to sunlight. It belongs to the family of disorders of the nucleotide excision repair system. Mutations of the ERCC6 and ERCC8 genes are the predominant cause of the Cockayne syndrome, whereby the ERCC6 gene mutation makes up approximately 70% of the cases. We report a Chinese case of a classic Cockayne syndrome, carrying the novel nonsense mutation c.1387C>T/Q463X in the ERCC6 gene in an apparently homozygous status. This mutation was found in a heterozygous status in this patients father, while the mother carried two wild-type ERCC6 alleles. A further molecular investigation of the family revealed that there was a de novo microdeletion including the ERCC6 gene of maternal origin in the proband. The determination of the deletion breakpoints by Illumina genome-wide DNA analysis beadchip showed that the deletion spanned 2.82 Mb in size. This case adds to the mutation spectrum of this DNA repair disorder.

Analysis of genetic mutations in Chinese patients with systemic primary carnitine deficiency.

Systemic primary carnitine deficiency (CDSP) is caused by mutations in SLC22A5 gene, which encodes organic cation transporter 2(OCTN2). CDSP leads to skeletal or cardiac myopathy and hepatic encephalopathy. The present study aimed to identify SLC22A5 gene mutations and analyze the potential relationship between genotype and clinical symptoms in 20 Chinese patients with CDSP. The complete coding region of the SLC22A5 gene including intron-exon boundaries were amplified and sequenced in all patients. Eighteen different mutations were found; of which, nine were novel. The mutations clustering in exons 1 and 4 accounted for 66.7% of all mutant alleles (26/39). The c.760C>T (p. R254X) was the most frequent mutation (25.6%, 10/39), suggesting it as an ethnic founder mutation. The relationship between genotype and phenotype was investigated in patients carrying the R254X mutation. Homozygous patients with R254X were late-onset cases who presented with dilated cardiomyopathy and muscle weakness after 1 year of age. Compound heterozygous patients carrying R254X, combined with other missense mutations occurred in very specific positions, dramatically altered OCTN2 protein function. Based on the analysis of case studies, a clear relationship between free carnitine (C0) level in plasma and OCTN2 genotype was not found in the present work, however, the low plasma C0 level could not indicate disease severity or genotype. Further functional studies with a large sample size are required to understand the relationship between R254X mutation and CDSP.

Clinical features and MUT gene mutation spectrum in Chinese patients with isolated methylmalonic acidemia: identification of ten novel allelic variants

BackgroundThis study aims to study MUT gene mutation spectrum in Chinese patients with isolated methylmalonic academia (MMA) and their clinical features for the potential genotype-phenotype correlation.MethodsForty-three patients were diagnosed with isolated MMA by elevated blood propionylcarnitine, propionylcarnitine to acetylcarnitine ratio, and urine methylmalonate without hyperhomocysteinemia. The MUT gene was amplified by polymerase chain reaction and directly sequenced. Those patients with at least one variant allele were included. The novel missense mutations were assessed by bioinformatic analysis and screened against alleles sequenced from 50 control participants.ResultsAmong the 43 patients, 38 had typical clinical presentations, and the majority (30/38) experienced earlyonset MMA. Eight patients died and seven were lost to follow-up. Twenty patients had poor outcomes and eight showed normal development. The 43 identified MUT gene mutations had at least one variant allele, whereas 35 had two mutant alleles. Of the 33 mutations reported before, eight recurrent mutations were identified in 32 patients, and c.729_730insTT (p.D244Lfs*39) was the most common (12/78) in the mutant alleles. Of the 10 novel mutations, six were missense mutations and four were premature termination codon mutations. The six novel missense mutations seemed to be pathogenic.ConclusionsA total of 10 novel MUT mutations were detected in the Chinese population. c.729_730insTT (p.D244Lfs*39) was the most frequent mutation. A genotype-phenotype correlation could not be found, but the genotypic characterization indicated the need of genetic counseling for MMA patients and early prenatal diagnoses for high-risk families.

Identification of five novel STAR variants in ten Chinese patients with congenital lipoid adrenal hyperplasia

Congenital lipoid adrenal hyperplasia (CLAH) is a rare autosomal recessive disorder caused by defective synthesis of all steroids. This disorder is characterized by 46,XY sex reversal, skin hyperpigmentation, early-onset adrenal crisis and enlarged adrenal with fatty accumulation. CLAH is caused by mutations in the STAR gene. The clinical features and STAR gene mutation spectrum of a large cohort of Chinese patients with CLAH were not reported previously. We performed clinical retrospective review and genetic analysis of the STAR gene in ten unrelated Chinese phenotypic female patients who were clinically diagnosed with CLAH and followed up in our hospital from 2006 to 2015. All ten patients, including two 46,XY females and eight 46,XX females, presented skin hyperpigmentation and early salt-wasting episode, and showed normal growth and development after steroid replacement treatment. Totally 20 mutant alleles containing 11 different STAR gene mutations were identified in these ten patients, including five novel variants (two missense and three null variants), all predicted to be pathogenic in bioinformatics analysis, and six mutations described in previous literature. Among these 11 mutations, a reported mutation c.772C>T and a novel variant c.707_708delinsCTT were most frequent, accounting for 35% and 15% of the total mutant alleles, respectively. This is the first report of a large Chinese cohort with CLAH, presenting the mutation spectrum of the STAR gene and two possible founder mutations in the Chinese population, which may contribute to better genetic counseling and prenatal diagnosis.

Mutation spectrum of hyperphenylalaninemia candidate genes and the genotype-phenotype correlation in the Chinese population

BACKGROUND Hyperphenylalaninemia (HPA) is an inherited metabolic disorder that is caused by a deficiency of phenylalanine hydroxylase (PAH) or tetrahydrobiopterin. The prevalence of HPA varies widely around the world. METHODS A spectrum of HPA candidate genes in 1020 Chinese HPA patients was reported. Sanger sequencing, next generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA) and quantitative real-time PCR (qRT-PCR) were applied to precisely molecular diagnose HPA patients. The allelic phenotype values (APV) and genotypic phenotype values (GPV) were calculated in PAH-deficient patients based on a recently developed formula. RESULTS Apart from genetic diagnoses confirmed in 915 HPA patients (89.7%) by Sanger sequencing, pathogenic variants were discovered in another 57 patients (5.6%) through deep detections (NGS, MLPA and qRT-PCR). We identified 196, 42, 10 and 2 variants in PAH, PTS, QDPR and GCH1, respectively. And a total of 47 novel variants were found in these genes. Through the APV and GPV calculations, it was found that the new GPV system was well correlated with metabolic phenotypes in most PAH-deficient patients. CONCLUSIONS More HPA candidate variants were identified using new molecular diagnostic methods. The new APV and GPV system is likely to be highly beneficial for predicting clinical phenotypes for PAH-deficient patients.

Chromosomal microarray analysis in developmental delay and intellectual disability with comorbid conditions

BackgroundDevelopmental delay (DD) and intellectual disability (ID) are frequently associated with a broad spectrum of additional phenotypes. Chromosomal microarray analysis (CMA) has been recommended as a first-tier test for DD/ID in general, whereas the diagnostic yield differs significantly among DD/ID patients with different comorbid conditions.MethodsTo investigate the genotype-phenotype correlation, we examined the characteristics of identified pathogenic copy number variations (pCNVs) and compared the diagnostic yields among patient subgroups with different co-occurring conditions.ResultsThis study is a retrospective review of CMA results generated from a mixed cohort of 710 Chinese patients with DD/ID. A total of 247 pCNVs were identified in 201 patients (28%). A large portion of these pCNVs were copy number losses, and the size of copy number losses was generally smaller than gains. The diagnostic yields were significantly higher in subgroups with co-occurring congenital heart defects (55%), facial dysmorphism (39%), microcephaly (34%) or hypotonia (35%), whereas co-occurring conditions of skeletal malformation (26%), brain malformation (24%) or epilepsy (24%) did not alter the yield. In addition, the diagnostic yield nominally correlated with ID severity.ConclusionVaried yields exist in DD/ID patients with different phenotypic presentation. The presence of comorbid conditions can be among factors to consider when planning CMA.