Yanru Huang

Targeted next-generation sequencing identifies novel compound heterozygous mutations of DYNC2H1 in a fetus with short rib-polydactyly syndrome, type III.

A 26-year-old woman with a past history of fetal skeletal dysplasia was referred to our institution at 24weeks of gestation following a routine sonographic diagnosis of short limbs in the fetus. A fetal ultrasound showed short limbs, a narrow thorax, short ribs with marginal spurs, and polydactyly. Conventional cytogenetics analysis of cultured amniocytes demonstrated that the fetal karyotype was normal. Using targeted exome sequencing of 226 known genes implicated in inherited skeletal dysplasia, we identified compound heterozygous mutations in the DYNC2H1 gene in the fetus with short rib-polydactyly syndrome, type III (SRPS III), c.1151 C>T(p.Ala384Val) and c.4351 C>T (p.Gln1451*), which were inherited from paternally and maternally, respectively. These variants were further confirmed using Sanger sequencing and have not been previously reported. To our knowledge, this is the first report of DYNC2H1 mutations causing SRPS III, in the Chinese population. Our findings expand the number of reported cases of this rare disease, and indicate that targeted next-generation sequencing (NGS) is an accurate, rapid, and cost-effective method in the genetic diagnosis of fetal skeletal dysplasia.

De novo exonic deletion of KDM6A in a Chinese girl with Kabuki syndrome: A case report and brief literature review

Kabuki syndrome (KS) is a rare condition with multiple congenital anomalies and mental retardation. Exonic deletions, disrupting the lysine (K)‐specific demethylase 6A (KDM6A) gene have been demonstrated as rare cause of KS. Here, we report a de novo 227‐kb deletion in chromosome Xp11.3 of a 7‐year‐old Chinese girl with KS. Besides the symptoms of KS, the patient also presented with skin allergic manifestations, which were considered to be a new, rare feature of the phenotypic spectrum. The deletion includes the upstream region and exons 1–2 of KDM6A and potentially causes haploinsuffiency of the gene. We also discuss the mutation spectrum of KDM6A and clinical variability of patients with KDM6A deletion through a literature review.

Novel variants in PAX6 gene caused congenital aniridia in two Chinese families

PurposeTo reveal the underlying genetic defect in two four-generation Chinese families with aniridia and explore the pathologic mechanism.MethodsFull ophthalmic examinations were performed in two families with aniridia. The PAX6 gene was directly sequenced in patients of two families, and the detected variants were screened in unaffected family members and two hundred unrelated healthy controls. Real-time quantitative PCR was used to explore pathologic mechanisms of the two variants.ResultsAniridia, cataract, and oscillatory nystagmus were observed in patients of the two families. In addition, we observed corneal opacity and microphthalmus in family 1, and strabismus, left ectopia lentis, microphthalmus, and microcornea in family 2. Sanger sequencing detected a novel 1-bp duplication (c.50dupA) in family 1 and a novel 2-bp splice site deletion (c.765+1_765+2delGT) in family 2. Sequencing of cDNA indicated skipping of exon 9 caused by the splice site deletion, being predicted to cause a premature stop codon, as well as the duplication. The PAX6 mRNA significantly lower in patients with aniridia than in unaffected family members in both families, suggesting that the duplication and splice site deletion caused nonsense-mediated mRNA decay.ConclusionsOur study identified two novel PAX6 variants in two families with aniridia and revealed the pathogenicity of the variants; this would expand the variant spectrum of PAX6 and help us better understand the molecular basis of aniridia, thus facilitating genetic counseling.

Two novel NIPBL gene mutations in Chinese patients with Cornelia de Lange syndrome.

Cornelia de Lange syndrome (CdLS) is a dominantly inherited developmental disorder characterized by distinctive facial features, mental retardation, and upper limb defects, with the involvement of multiple organs and systems. To date, mutations have been identified in five genes responsible for CdLS: NIPBL, SMC1A, SMC3, RAD21, and HDAC8. Here, we present a clinical and molecular characterization of five unrelated Chinese patients whose clinical presentation is consistent with that of CdLS. There were no chromosomal abnormalities in the five children. In three patients, DNA sequencing revealed a previously reported frameshift mutation c.2479delA (p.Arg827GlyfsX20), and two novel mutations including a heterozygous mutation c.6272 G>T (p.Cys2091Phe) and a frameshift mutation c.1672delA (p.Thr558LeufsX7) in NIPBL. For the remaining patients, large deletions and/or duplications within the NIPBL gene were excluded as playing a role in the pathogenesis, by Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. These findings broaden the mutation spectrum of NIPBL and further our understanding of the diverse and variable effects of NIPBL mutations on CdLS.

A novel deletion mutation in RS1 gene caused X-linked juvenile retinoschisis in a Chinese family.

PurposeX-linked juvenile retinoschisis (XLRS), a leading cause of juvenile macular degeneration, is characterized by a spoke-wheel pattern in the macular region of the retina and splitting of the neurosensory retina. This study aimed to identify the underlying genetic defect in a Chinese family with XLRS.MethodsThe proband underwent complete ophthalmic examinations, including fundus examination, fundus autofluorescence, and optical coherence tomography. DNA extracted from proband and his younger brother was screened for mutations in RS1 gene. The detected RS1 mutation was tested in all available family members and 200 healthy controls.ResultsReduced visual acuity, spoke-wheel pattern at the fovea, and split retina were observed in the proband. A novel frameshift mutation c.206-207delTG in the RS1 gene, leading to a truncated protein (p.L69fs16X), was identified in the proband and his younger brother. This mutation was not found in any unaffected member or in the healthy controls. The mother of the proband was hemizygous for this mutant allele.ConclusionsWe identified a novel causative mutation of RS1 in a Chinese family with XLRS. This finding expands the mutation spectrum of RS1 and provides evidence for a phenotype–genotype study in XLRS.

Identification of a novel MIP frameshift mutation associated with congenital cataract in a Chinese family by whole-exome sequencing and functional analysis

PurposeTo detect the underlying pathogenesis of congenital cataract in a four-generation Chinese family.MethodsWhole-exome sequencing (WES) of family members (III:4, IV:4, and IV:6) was performed. Sanger sequencing and bioinformatics analysis were subsequently conducted. Full-length WT-MIP or K228fs-MIP fused to HA markers at the N-terminal was transfected into HeLa cells. Next, quantitative real-time PCR, western blotting and immunofluorescence confocal laser scanning were performed.ResultsThe age of onset for nonsyndromic cataracts in male patients was by 1-year old, earlier than for female patients, who exhibited onset at adulthood. A novel c.682_683delAA (p.K228fs230X) mutation in main intrinsic protein (MIP) cosegregated with the cataract phenotype. The instability index and unfolded states for truncated MIP were predicted to increase by bioinformatics analysis. The mRNA transcription level of K228fs-MIP was reduced compared with that of WT-MIP, and K228fs-MIP protein expression was also lower than that of WT-MIP. Immunofluorescence images showed that WT-MIP principally localized to the plasma membrane, whereas the mutant protein was trapped in the cytoplasm.ConclusionsOur study generated genetic and primary functional evidence for a novel c.682_683delAA mutation in MIP that expands the variant spectrum of MIP and help us better understand the molecular basis of cataract.

A lesson from a reported pathogenic variant in Peutz-Jeghers syndrome: a case report

Peutz-Jeghers syndrome (PJS) is a rare autosomal dominant disorder characterized by mucocutaneous hyperpigmentation, gastrointestinal (GI) hamartmatous polyps, and an increased risk of various malignancies. Pathogenic variants in the LKB1 tumor suppressor gene (also known as STK11) are the major cause of PJS. In this study, compound heterozygous variants of LKB1, c.890G > A/ c.1062C > G and del(exon1)/ c.1062C > G, were identified in two sporadic Chinese PJS cases respectively. Although all these three variants had been related to the autosomal dominant PJS in previous studies, all evidences collected in this study including de novo data, segregation data, population data, in-silico data, and functional data indicated that del(exon1) and c.890G > A are pathogenic in these two PJS families rather than c.1062C > G. This finding would contribute to genetic counseling for individuals carrying the variant c.1062C > G with or without PJS phenotypes. Moreover, this finding reminds genetic counselors that it is necessary to reevaluate the pathogenicity of reported variants in a known Mendelian disorder in order to avoid a misleading decision.

A novel MSX1 intronic mutation associated with autosomal dominant non-syndromic oligodontia in a large Chinese family pedigree.

BACKGROUND Tooth agenesis is a common developmental dental anomaly. The aim of the study was to identify the causal genetic mutation in a four-generation Chinese family affected with non-syndromic autosomal dominant tooth agenesis. METHODS Genome-wide scanning was performed using the Illumina Linkage-12 array. Genotyping of short tandem repeat markers was used to finely map the causative locus. Haplotype analysis and Sanger sequencing was performed to precisely locate the position and nature of the gene defect. RESULTS Clinical examination of the available 23 family members showed variable tooth agenesis in 10 subjects, ranging from oligodontia to mild hypodontia. Genome-wide scanning and haplotype analyses identified the 4p16.1-p16.3 region with a maximum multi-point LOD score of 3.50, which overlapped with the MSX1 gene. A single heterozygous point mutation IVS1-5 G>A in the MSX1 gene was exclusively detected in the 10 family members affected with tooth agenesis. Sequencing of MSX1 cDNA revealed that the intronic mutation did not affect the normal splicing pattern of the pre-mRNA. However, real-time qPCR analysis of lymphocyte RNA showed that the level of MSX1 mRNA was significantly decreased in individuals heterozygous for the mutation. CONCLUSIONS We identified and characterized a novel intronic mutation in the MSX1 gene in a large Chinese pedigree, adding to the small repertoire of MSX1 mutations associated with autosomal dominant tooth agenesis. We hypothesize that the variable degree of tooth agenesis observed in each affected individual may be due to sub-optimal levels of MSX1 expression during critical stages tooth development.

Morphometric analysis and developmental comparison of embryos from carriers with balanced chromosomal rearrangements in preimplantation genetic diagnosis cycles

The morphological parameters of embryos from 22 carriers with balanced chromosomal rearrangements (CRs) were quantified and evaluated to determine their possible link to chromosomal composition. The morphometric characteristics of 168 embryos diagnosed by fluorescence in situ hybridisation were measured using an imaging tool and then analysed retrospectively. The mean zygotic diameter of normal-balanced embryos was significantly smaller compared with that of abnormal embryos (P=0.015). In addition, the reduction in total cytoplasmic volume for Day-3 embryos was significantly lower in normal or balanced embryos than in abnormal embryos (P=0.027). Moreover, the pronuclear volumes of embryos that failed to reach the blastocyst stage were significantly smaller compared with those of blastocysts (P=0.016). These findings indicate that morphometric characteristics are correlated with developmental outcomes as well as with chromosomal composition in embryos from balanced CR carriers. However, an effective indicator of developmental outcomes may not accurately reflect chromosomal composition. Combining morphometric and traditional qualitative assessment may increase the precision and standardisation of embryo evaluation as well as contributing to improved efficiency of preimplantation genetic diagnosis by selecting embryos with high developmental potential and preferentially testing embryos predicted to have a low risk of chromosomal imbalance.

Novel de novo nonsense mutation of the PHEX gene (p.Lys50Ter) in a Chinese patient with hypophosphatemic rickets

X-linked hypophosphatemic rickets (XLHR), the most common form of inherited rickets, is a dominant disorder characterized by hypophosphatemia, abnormal bone mineralization, and short stature. Mutations in the PHEX gene are major causes of XLHR. Herein, we clinically characterized four unrelated families with hypophosphatemia, bone abnormalities, short stature, and dentin malformation. Mutational analysis of the PHEX gene using Sanger sequencing revealed three recurrent mutations (c.2197T>C, c.1646G>C, and c.2198G>A) and a de novo nonsense mutation (c.148A>T). The novel mutation was not found in any of the unaffected family members or in the 100 healthy controls and was predicted to produce a truncated protein (p.K50X), a truncated form of the PHEX protein caused by nonsense mutations has been frequently detected in XLHR individuals. Thus, our work indicated that the c.148A>T (p.K50X) mutation was the likely pathogenic mutation in individual III-2 in family 2, and that PHEX gene mutations were responsible for XLHR in these Chinese families. These findings expand the mutation spectrum of PHEX and may help us to understand the molecular basis of XLHR in order to facilitate genetic counseling.