Qihua Fu

Investigation of somatic NKX2-5, GATA4 and HAND1 mutations in patients with tetralogy of Fallot.

Aims: Tetralogy of Fallot (TOF) is the most common type of congenital heart disease (CHD). Several genes essential for heart development have been identified. In recent years, there have been a few reports of the high frequency of somatic mutation in the heart tissues of CHD patients. The majority of findings were reported by the same investigators using formalin fixed tissues. In this study, we investigate the possibility of somatic mutation in fresh pathological cardiac tissues. Methods: We sequenced all exons and their boundaries of the NKX2-5, GATA4 and HAND1 genes from the right ventricular outflow tract muscle and peripheral blood of 38 unrelated non-syndromic TOF children who had undergone routine surgery. The same group of genes was also sequenced in the peripheral blood of 105 control subjects. We also compared the DNA yield in different tissue storage methods and sequenced 10 formalin fixed specimens. Results: Sequencing cardiac muscle and peripheral blood from the same patient showed identical findings. The previously reported p.Pro407Gln mutation in GATA4 gene and a novel heterozygous p.Ala58Glu mutation in HAND1 gene were identified in two of the 38 patients, both in the affected cardiac tissues and in the lymphocytes. These mutations were not seen in any control subjects. The formalin fixation rendered less DNA yield than the frozen method, but no artificial mutations were generated. Conclusions: Our study shows no evidence of somatic NKX2-5, GATA4 and HAND1 mutations playing a role in the pathogenesis of TOF. Our findings suggest that the GATA4 and HAND1 germline mutations are associated with non-syndromic CHD.

The Fas/Fas Ligand Death Receptor Pathway Contributes to Phenylalanine-Induced Apoptosis in Cortical Neurons

Phenylketonuria (PKU), an autosomal recessive disorder of amino acid metabolism caused by mutations in the phenylalanine hydroxylase (PAH) gene, leads to childhood mental retardation by exposing neurons to cytotoxic levels of phenylalanine (Phe). A recent study showed that the mitochondria-mediated (intrinsic) apoptotic pathway is involved in Phe-induced apoptosis in cultured cortical neurons, but it is not known if the death receptor (extrinsic) apoptotic pathway and endoplasmic reticulum (ER) stress-associated apoptosis also contribute to neurodegeneration in PKU. To answer this question, we used specific inhibitors to block each apoptotic pathway in cortical neurons under neurotoxic levels of Phe. The caspase-8 inhibitor Z-IETD-FMK strongly attenuated apoptosis in Phe-treated neurons (0.9 mM, 18 h), suggesting involvement of the Fas receptor (FasR)-mediated cell death receptor pathway in Phe toxicity. In addition, Phe significantly increased cell surface Fas expression and formation of the Fas/FasL complex. Blocking Fas/FasL signaling using an anti-Fas antibody markedly inhibited apoptosis caused by Phe. In contrast, blocking the ER stress-induced cell death pathway with salubrinal had no effect on apoptosis in Phe-treated cortical neurons. These experiments demonstrate that the Fas death receptor pathway contributes to Phe-induced apoptosis and suggest that inhibition of the death receptor pathway may be a novel target for neuroprotection in PKU patients.

Molecular defects identified by whole exome sequencing in a child with Fanconi anemia.

Fanconi anemia is a rare genetic disease characterized by bone marrow failure, multiple congenital malformations, and an increased susceptibility to malignancy. At least 15 genes have been identified that are involved in the pathogenesis of Fanconi anemia. However, it is still a challenge to assign the complementation group and to characterize the molecular defects in patients with Fanconi anemia. In the current study, whole exome sequencing was used to identify the affected gene(s) in a boy with Fanconi anemia. A recurring, non-synonymous mutation was found (c.3971C>T, p.P1324L) as well as a novel frameshift mutation (c.989_995del, p.H330LfsX2) in FANCA gene. Our results indicate that whole exome sequencing may be useful in clinical settings for rapid identification of disease-causing mutations in rare genetic disorders such as Fanconi anemia.

Exome sequencing reveals a novel PTHLH mutation in a Chinese pedigree with brachydactyly type E and short stature.

Brachydactyly includes shortening of digits due to abnormal development of phalanges, metacarpals, or both. It can occur either as an isolated malformation or with other anomalies as part of many congenital syndromes. It is included as one of the dysostosis groups affecting the limbs in the nosology and classification of genetic skeletal disorders. However, brachydactyly usually shows a high degree of phenotypic variability. In this study, we successfully identified a novel heterozygous mutation of the parathyroid hormone-like hormone (PTHLH) gene by exome sequencing in a Chinese pedigree with brachydactyly and short stature. The PTHLH gene encodes a parathyroid hormone-related protein (PTHrP) that is involved in the regulation of endochondral bone development, and mutations in this gene cause the type E form of brachydactyly. The mutation p.L15R occurs at a hydrophobic core region of the signal peptide, suggesting that this variation probably changes the signal peptide cleavage site at the in silico prediction. Further in vitro functional analysis showed that this mutation can lead to the retention of an N-terminal signal peptide fragment after the nascent proteins are translated.

Novel frame-shift mutations of GLI3 gene in non-syndromic postaxial polydactyly patients

Polydactyly is a common congenital limb deformity. This anomaly may occur in isolation (non-syndromic) or as part of a syndrome. The glioma-associated oncogene family zinc finger 3 (GLI3) is known to be associated with both syndromic and non-syndromic polydactyly. GLI3 plays a predominant role in the pathogenesis of syndromic polydactyly: mutations have been identified in 68% of patients with Greig cephalopolysyndactyly syndrome and 91% of patients with Pallister-Hall syndrome. The knowledge regarding the contribution of GLI3 in non-syndromic polydactyly is currently very limited. In this study, we assembled a cohort of individuals of Chinese ethnicity with non-syndromic postaxial polydactyly. We presented the clinical features and molecular evaluations of 19 probands. GLI3 mutations were identified in 15.8% of probands (3/19) including two novel frame-shift mutations c.3855dupC (p.Met1286HisfsTer18) and c.4141delA (p.Arg1381GlyfsTer38) detected in sporadic cases and one previously reported nonsense mutation (c.1927C>T/p.Arg643Ter) in a familial case. Of note, GLI3 mutations were exclusively detected in patients with bilateral polydactyly affecting both hands and feet. Three out of five (60%) probands with bilateral polydactyly on both hands and feet carried pathogenic mutations in GLI3. Our study demonstrated the role of GLI3 in a significant fraction of patients with non-syndromic bilateral polydactyly affecting both hands and feet.

A Novel Missense Mutation of GATA4 in a Chinese Family with Congenital Heart Disease.

Background Congenital heart disease (CHD) is the most prevalent type of birth defect in human, with high morbidity in infant. Several genes essential for heart development have been identified. GATA4 is a pivotal transcription factor that can regulate the cardiac development. Many GATA4 mutations have been identified in patients with different types of CHD. Aims In this study, the NKX2-5, HAND1 and GATA4 coding regions were sequenced in a family spanning three generations in which seven patients had CHD. Disease-causing potential variation in this family was evaluated by bioinformatics programs and the transcriptional activity of mutant protein was analyzed by the dual luciferase reporter assay. Results A novel GATA4 mutation, c.C931T (p.R311W), was identified and co-segregated with the affected patients in this family. The bioinformatics programs predicted this heterozygous mutation to be deleterious and the cross-species alignment of GATA4 sequences showed that the mutation occurred within a highly conserved amino acid. Even though it resided in the nuclear localization signal domain, the mutant protein didn’t alter its intracellular distribution. Nevertheless, further luciferase reporter assay demonstrated that the p.R311W mutation reduced the ability of GATA4 to activate its downstream target gene. Conclusions Our study identified a novel mutation in GATA4 that likely contributed to the CHD in this family. This finding expanded the spectrum of GATA4 mutations and underscored the pathogenic correlation between GATA4 mutations and CHD.

Identification of three FGA mutations in two Chinese families with congenital afibrinogenaemia

Summary.  Congenital afibrinogenaemia is a rare autosomal recessive disorder, characterized by the complete absence or extremely reduced level of fibrinogen (Fg). We attempted to analyse the phenotype and genotype in two Chinese families with congenital afibrinogenaemia. Coagulation studies including activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) and Fg were performed in the patients and other family members. All the exons, exon–intron boundaries and promoter regions of three Fg genes (FGA, FGB and FGG) were screened by direct sequencing. Three patients in two families suffered from moderate to severe haemorrhage. Their APTT, PT and TT were extremely prolonged and plasma Fg levels were undetectable by Clauss method and extremely reduced by immunoassay. Genetic analysis revealed three FGA mutations in three patients including one novel mutation. In family 1, patient 1 was detected compound heterozygous mutations in FGA, g.1892–1899delAGTA/GTAA from her patriline and g.1978‐g.3215del1238 bp from her matriline. In family 2, a homozygous Gln203X in Aα‐chain was found in both patients 2 and 3 due to consanguineous marriage. All these mutations were null mutations, which could produce premature stop codons in FGA. It can be indicated that with more genetic analysis performed on afibrinogenaemia patients all over the world, there is no distinct difference in geographical distribution of Fg gene mutations. Gln203X in Aα‐chain was first reported in this study, which may help to further understand the function of Aα‐chain.

A novel mutation in NPHS2 gene identified in a Chinese pedigree with autosomal recessive steroid-resistant nephrotic syndrome

Aims: Steroid‐resistant nephrotic syndrome (SRNS) is an inherent deficiency of podocyte caused by mutations of genes encoding slit diaphragm proteins. Mutations in NPHS2, encoding podocin, have been identified as responsible for childhood‐onset familial SRNS. The present study revealed the genotype of a Chinese pedigree with autosomal recessive (AR) SRNS and reported a novel disease‐causing NPHS2 mutation. Methods: A Chinese pedigree with AR‐SRNS was enrolled in the study. All eight exons and exon‐intron boundaries of NPHS2 genes were amplified from the genomic DNA of the family members and analysed by direct sequencing. The deficient expression of the mutant protein was illustrated by indirect immunofluorescence. Results: A compound heterozygous NPHS2 mutation (c.211C > T /c.460dupT) was found in the proband. The paternal c.211C > T is a novel point mutation, resulting in an immediate stop codon (p.Arg71X). The maternal c.460dupT is a frameshift mutation introducing an earlier stop codon (p.Phe156AspfsX10). Both mutations could be expected to lead to truncated protein of podocin. Abnormal expression and distribution of the mutated protein were also exhibited in the patient. Conclusions: The compound heterozygous mutation in NPHS2 may explain the development of SRNS in this family. p.Arg71X is a novel disease‐causing mutation leading to a deficient expression of podocin.

Three novel missense mutations in the filamin B gene are associated with isolated congenital talipes equinovarus

Congenital talipes equinovarus (CTEV) is one of the most common musculoskeletal disorders. Genetic factors have been suggested to be an important contributor to its pathogenesis. Some genes, including PITX1, TBX4, and RBM10, have been associated with CTEV. We aimed to determine the disease-causing mutations in Chinese patients with isolated CTEV. Genomic DNA was extracted from peripheral blood samples of a three-generation pedigree and 53 sporadic patients with CTEV. Whole-exome sequencing and Sanger sequencing were used to identify and validate disease-causing mutations, respectively. A putative pathogenic mutation c.4717G>T (p.D1573Y) in the filamin B (FLNB) gene, which co-segregated with CETV, was identified in the pedigree. Two additional novel missense mutations in the same gene [c.1897A>G (p.M633V) and c.2195A>G (p.Y732C)] were identified from the 53 sporadic patients. Plasmids expressing wild-type or mutant constructs were transfected into HEK293T cells to determine whether these amino acid substitutions affect protein activity. All three (M633V, Y732C, and D1573Y) affected FLNB protein expression and led to cytoplasmic focal accumulation. Our results provide evidence for the involvement of FLNB in the pathogenesis of isolated CTEV and have expanded the clinical spectrum of FLNB mutations.

Novel Mutations in the SCNN1A Gene Causing Pseudohypoaldosteronism Type 1

Pseudohypoaldosteronism type 1 (PHA1) is a rare inherited disease characterized by resistance to the actions of aldosterone. Mutations in the subunit genes (SCNN1A, SCNN1B, SCNN1G) of the epithelial sodium channel (ENaC) and the NR3C2 gene encoding the mineralocorticoid receptor, result in systemic PHA1 and renal PHA1 respectively. Common clinical manifestations of PHA1 include salt wasting, hyperkalaemia, metabolic acidosis and elevated plasma aldosterone levels in the neonatal period. In this study, we describe the clinical and biochemical manifestations in two Chinese patients with systemic PHA1. Sequence analysis of the SCNN1A gene revealed a compound heterozygous mutation (c.1311delG and c.1439+1G>C) in one patient and a homozygous mutation (c.814_815insG) in another patient, all three variants are novel. Further analysis of the splicing pattern in a minigene construct showed that the c.1439+1G>C mutation can lead to the retainment of intron 9 as the 5′-donor splice site disappears during post-transcriptional processing of mRNA. In conclusion, our study identified three novel SCNN1A gene mutations in two Chinese patients with systemic PHA1.