Zhaojing Zheng

Chromosome microarray testing for patients with congenital heart defects reveals novel disease causing loci and high diagnostic yield

BackgroundCongenital heart defects (CHD), as the most common congenital anomaly, have been reported to be frequently associated with pathogenic copy number variants (CNVs). Currently, patients with CHD are routinely offered chromosomal microarray (CMA) testing, but the diagnostic yield of CMA on CHD patients has not been extensively evaluated based on a large patient cohort. In this study, we retrospectively assessed the detected CNVs in a total of 514 CHD cases (a 422-case clinical cohort from Boston Childrens Hospital (BCH) and a 92-case research cohort from Shanghai Children’s Medical Center (SCMC)) and conducted a genotype-phenotype analysis. Furthermore, genes encompassed in pathogenic/likely pathogenic CNVs were prioritized by integrating several tools and public data sources for novel CHD candidate gene identification.ResultsBased on the BCH cohort, the overall diagnostic yield of CMA testing for CHD patients was 12.8(pathogenic CNVs)-18.5% (pathogenic and likely pathogenic CNVs). The diagnostic yield of CMA for syndromic CHD was 14.1-20.6% (excluding aneuploidy cases), whereas the diagnostic yield for isolated CHD was 4.3-9.3%. Four recurrent genomic loci (4q terminal region, 15q11.2, 16p12.2 and Yp11.2) were more significantly enriched in cases than in controls. These regions are considered as novel CHD loci. We further identified 20 genes as the most likely novel CHD candidate genes through gene prioritization analysis.ConclusionThe high clinical diagnostic yield of CMA in this study provides supportive evidence for CMA as the first-line genetic diagnostic tool for CHD patients. The CNVs detected in our study suggest a number of CHD candidate genes that warrant further investigation.

Screening for Coding Variants in FTO and SH2B1 Genes in Chinese Patients with Obesity

Objective To investigate potential functional variants in FTO and SH2B1 genes among Chinese children with obesity. Methods Sanger sequencing of PCR products of all FTO and SH2B1 exons and their flanking regions were performed in 338 Chinese Han children with obesity and 221 age- and sex-matched lean controls. Results A total of seven and five rare non-synonymous variants were identified in FTO and SH2B1, respectively. The overall frequencies of FTO and SH2B1 rare non-synonymous variants were similar in obese and lean children (2.37% and 0.90% vs. 1.81% and 1.36%, P>0.05). However, four out of the seven variants in FTO were novel and all were unique to obese children (p>0.05). None of the novel variants was consistently being predicted to be deleterious. Four out of five variants in SH2B1 were novel and one was unique to obese children (p>0.05). One variant (L293R) that was consistently being predicted as deleterious in SH2B1 gene was unique to lean control. While rare missense mutations were more frequently detected in girls from obesity as well as lean control than boys, the difference was not statistically significant. In addition, its shown that the prevalence of rare missense mutations of FTO as well as SH2B1 was similar across different ethnic groups. Conclusion The rare missense mutations of FTO and SH2B1 did not confer risks of obesity in Chinese Han children in our cohort.

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.

A New Subtype of Multiple‐Synostoses Syndrome is Caused by a Mutation in GDF6 that Decreases its Sensitivity to Noggin and Enhances its Potency as a BMP Signal

Growth and differentiation factors (GDFs) are secreted signaling molecules within the BMP family that have critical roles in joint morphogenesis during skeletal development in mice and humans. Using genetic data obtained from a six‐generation Chinese family, we identified a missense variant in GDF6 (NP_001001557.1; p.Y444N) that fully segregates with a novel autosomal dominant synostoses (SYNS) phenotype, which we designate as SYNS4. Affected individuals display bilateral wrist and ankle deformities at birth and progressive conductive deafness after age 40 years. We find that the Y444N variant affects a highly conserved residue of GDF6 in a region critical for binding of GDF6 to its receptor(s) and to the BMP antagonist NOG, and show that this mutant GDF6 is a more potent stimulator of the canonical BMP signaling pathway compared with wild‐type GDF6. Further, we determine that the enhanced BMP activity exhibited by mutant GDF6 is attributable to resistance to NOG‐mediated antagonism. Collectively, our findings indicate that increased BMP signaling owing to a GDF6 gain‐of‐function mutation is responsible for loss of joint formation and profound functional impairment in patients with SYNS4. More broadly, our study highlights the delicate balance of BMP signaling required for proper joint morphogenesis and reinforces the critical role of BMP signaling in skeletal development.

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.

Identification of LDLR mutations in two Chinese pedigrees with familial hypercholesterolemia

Abstract Familial hypercholesterolemia (FH) is an autosomal dominant, inherited disease (OMIM 143890) characterized by elevated serum cholesterol bound to low-density lipoprotein (LDL). It is mainly caused by mutations of the low-density lipoprotein receptor gene (LDLR). In this study, we investigated two Chinese pedigrees with FH. The probands were a 9-year-old boy and a 1-year-old boy, who had high LDL-C levels. The proband in family A showed skin xanthoma. We sequenced the promoter and all exons and exon-intron boundaries of the LDLR gene to detect potential mutations. Compound heterozygote of c.1747C>T and c.2054C>T was detected in the proband of family A, and a heterozygous indel mutation c.551_553 delGTAinsTT was discovered in the second family. The c.1747C>T and c.2054C>T mutations, which have been reported previously, result in His583Tyr and Pro685Leu substitutions, respectively. The novel c.551_553 delGTAinsTT indel mutation causes a frameshift, which results in a p.Cys184Phe fs21X mutation in the corresponding protein.

A novel missense mutation of TNNI2 in a Chinese family cause distal arthrogryposis type 1.

The distal arthrogryposis (DA) syndromes are a group of disorders characterized by congenital contractures of limbs. According to phenotypical characteristics, DA syndromes have been clinically classified into 10 types. Currently, at least nine disease causing genes have been identified for different types of DA. Here, we report a 3‐generation Chinese pedigree with three DA affected members. We performed whole exome sequencing on two affected and one unaffected individuals of this family and successfully identified a novel missense mutation in TNNI2 as the pathogenic mutation. The TNNI2 gene encodes a subunit of the troponin complex, a contractile machinery of the muscle. The mutation p.F178C that could change the H‐bond formation of a neighboring residue occurs at a highly conserved position, suggesting that this variation probably affects the TNNI2 protein function. Our study also demonstrates the power of whole exome sequencing in causal mutation identification for phenotypically variable and genetically heterogeneous disorders.

Two novel isovaleryl-CoA dehydrogenase gene mutations in a Chinese infant

Isovaleric acidemia (IVA) is a rare inherited metabolic disease caused by a deficiency in isovaleryl-CoA dehydrogenase (IVD). Newborn screening with tandem mass spectrometry leads to early identification of individuals with risk of IVA. The family specific mutations are useful for prenatal diagnosis. Molecular genetic analysis helps to further confirm the clinical diagnosis of IVA. We describe here the clinical and metabolic features of a Chinese infant with early onset IVA. Sequence analysis of the IVD gene identifies compound heterozygous mutations in this patient, c.39G>A (p.W13X) nonsense mutation and c.597C>G (p.I199 M) missense mutation, both of which are previously unreported. Structural analyses suggest that the p.I199 M missense mutation may destabilize the IVD monomer structure and affect the interaction between IVD and flavin adenine dinucleotide. Both the clinical and genetic features of this patient help to further expand our knowledge of IVA.

A unique combination of 17pter trisomy and 21qter monosomy in a boy with developmental delay, severe intellectual disability, growth retardation and dysmorphisms.

BACKGROUND Microduplication at 17p13.3 and microdeletion at 21q22 are both rare chromosomal aberrations. The presence of both genomic imbalances in one patient has not been previously reported in literature. In this study, we performed a molecular diagnostic testing with a whole genome microarray on a 3-year-old boy with developmental delay, mental retardation and multiple malformations. METHODS A routine G-banding karyotype analysis was performed using peripheral lymphocytes. Chromosome microarray analysis (CMA) was done using Affymetrix CytoScan™ HD array. Genomic imbalances were further confirmed by multiple ligation-dependent probe amplification (MLPA). RESULTS The result of karyotyping was normal but CMA detected a 9.8 Mb microduplication at 17p13.3-13.1 (chr17: 1-9,875,545) and a 2.8 Mb microdeletion involving 21q22.3-qter (chr21: 45,239,077-48,097,372). The imbalances were due to a balanced translocation present in patients mother. The patient was characterized with short stature, profound developmental delay, non-verbal, intellectual disability as well as craniofacial dysmorphism, subtle brain structural anomaly and sparse scalp hair. CONCLUSIONS This is the first patient reported with a combination of a microduplication at 17p13.3-13.1 and a microdeletion at 21q22.3-qter. Both genomic imbalances were undetected by conventional karyotyping but were delineated with CMA test. Synergistic effect from the two rare genomic imbalances is likely responsible for the severe clinical phenotypes observed in this patient.

A novel GJA1 mutation identified by whole exome sequencing in a Chinese family with autosomal dominant syndactyly

Abstract Syndactyly is one of the most common hereditary limb malformations characterized by fusion of adjacent fingers and/or toes. The current classification scheme of non-syndromic syndactyly defines at least nine well-characterized syndactylous entities with subdivisions based on phenotype and genotype. Here, we reported a 3-generation Chinese pedigree with four affected Syndactyly members inherited in an autosomal dominant manner. We performed whole exome sequencing on two affected members and successfully identified a novel missense mutation p.R101L in GJA1 as the pathogenic mutation. The manifestations caused by GJA1 R101L mutation are different from typical characteristics of oculodentodigital dysplasia. Connexin 43 (Cx43), encoded by GJA1, plays a key role in normal facial and limb development. Arg101Leu mutation caused a hydrophobic to hydrophilic substitution, changing the structural integrity and stability of the molecule. Three-dimensional structural analysis showed this mutation could alter the conformation of residue side chain and produce steric clashes with spatial adjacent residues and cause folding destabilization, suggesting this amino acid appears to play an important role in the structure and function of Cx43. Our study also demonstrates the power of whole exome sequencing in causal mutation identification for phenotypically variable and genetically heterogeneous disorders.