Haoxian Li

Non‐invasive prenatal testing of fetal whole chromosome aneuploidy by massively parallel sequencing

To determine whether non‐invasive prenatal testing by maternal plasma DNA sequencing can uncover all fetal chromosome aneuploidies in one simple sequencing event.

Noninvasive Prenatal Testing for Wilson Disease by Use of Circulating Single-Molecule Amplification and Resequencing Technology (cSMART)

BACKGROUND Noninvasive prenatal testing (NIPT) for monogenic diseases by use of PCR-based strategies requires precise quantification of mutant fetal alleles circulating in the maternal plasma. The study describes the development and validation of a novel assay termed circulating single-molecule amplification and resequencing technology (cSMART) for counting single allelic molecules in plasma. Here we demonstrate the suitability of cSMART for NIPT, with Wilson Disease (WD) as proof of concept. METHODS We used Sanger and whole-exome sequencing to identify familial ATP7B (ATPase, Cu(++) transporting, β polypeptide) gene mutations. For cSMART, single molecules were tagged with unique barcodes and circularized, and alleles were targeted and replicated by inverse PCR. The unique single allelic molecules were identified by sequencing and counted, and the percentage of mutant alleles in the original maternal plasma sample was used to determine fetal genotypes. RESULTS Four families with WD pedigrees consented to the study. Using Sanger and whole-exome sequencing, we mapped the pathogenic ATP7B mutations in each pedigree and confirmed the probands original diagnosis of WD. After validation of cSMART with defined plasma models mimicking fetal inheritance of paternal, maternal, or both parental mutant alleles, we retrospectively showed in second pregnancies that the fetal genotypes assigned by invasive testing and NIPT were concordant. CONCLUSIONS We developed a reliable and accurate NIPT assay that correctly diagnosed the fetal genotypes in 4 pregnancies at risk for WD. This novel technology has potential as a universal strategy for NIPT of other monogenic disorders, since it requires only knowledge of the parental pathogenic mutations.

Copy Number Variation Sequencing for Comprehensive Diagnosis of Chromosome Disease Syndromes

Detection of chromosome copy number variation (CNV) plays an important role in the diagnosis of patients with unexplained clinical symptoms and for the identification of chromosome disease syndromes in the established fetus. In current clinical practice, karyotyping, in conjunction with array-based methods, is the gold standard for detection of CNV. To increase accessibility and reduce patient costs for diagnostic CNV tests, we speculated that next-generation sequencing methods could provide a similar degree of sensitivity and specificity as commercial arrays. CNV in patient samples was assessed on a medium-density single nucleotide polymorphism array and by low-coverage massively parallel CNV sequencing (CNV-seq), with mate pair sequencing used to confirm selected CNV deletion breakpoints. A total of 10 ng of input DNA was sufficient for accurate CNV-seq diagnosis, although 50 ng was optimal. Validation studies of samples with small CNVs showed that CNV-seq was specific and reproducible, suggesting that CNV-seq may have a potential genome resolution of approximately 0.1 Mb. In a blinded study of 72 samples with known gross and submicroscopic CNVs originally detected by single nucleotide polymorphism array, there was high diagnostic concordance with CNV-seq. We conclude that CNV-seq is a viable alternative to arrays for the diagnosis of chromosome disease syndromes.

Phenotypic expansion of the interstitial 16p13.3 duplication: a case report and review of the literature.

Genotype-phenotype analysis of at least 25 individuals with interstitial 16p13.3 duplications defines a recognizable syndrome associated with duplication of a critical Rubinstein-Taybi region encompassing only the CREBBP gene. Nevertheless, variable or incompletely penetrant phenotype has been reported previously. We here report a case of a 5-year old boy with a recognizable phenotype of this syndrome, including intellectual disability, mild arthrogryposis, small and proximally implanted thumbs and characteristic facial features. In addition, growth delay, microcephaly and distinguishable structural brain MRI abnormalities were observed. A de novo 1.5 Mb interstitial duplication of 16p13.3 was detected by SNP-array and fluorescence in situ hybridization (FISH). Short tandem repeat polymorphism (STRP) analysis with marker D16S475 indicated that the duplication was formed before maternal meiosis II. Our findings highlight the variable clinical features and further expand the phenotypic spectrum correlated with this lately proposed syndrome.

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.

Rare intracranial cholesterol deposition and a homozygous mutation of LDLR in a familial hypercholesterolemia patient.

Familial hypercholesterolemia (FH MIM# 143890) is one of the most common autosomal inherited diseases. FH is characterized by elevated plasma levels of total cholesterol and low-density lipoprotein-cholesterol. Mutation in the LDLR gene, which encodes the LDL receptor protein, is responsible for most of the morbidity of FH. The incidence of heterozygous FH is about 1/500, whereas the incidence of homozygous FH is only 1/1,000,000 in Caucasian population. In this study, we report a homozygous LDLR mutation (c.298G>A) in a familial hypercholesterolemia patient, who exhibited intracranial cholesterol deposition, which is a rare addition to the common FH phenotypes. The probands consanguineous parents have the same heterozygous mutation with elevated concentrations of LDL-C but no xanthoma.

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.

Clinical and molecular investigation in Chinese patients with glutaric aciduria type I.

Glutaric aciduria type I (GA-I) is a rare autosomal recessive metabolic disorder caused by deficiency of glutaryl-CoA dehydrogenase (GCDH), leading to an abnormal metabolism of lysine, hydroxylysine and tryptophan. It results in accumulations of glutaric acid, 3-hydroxyglutaric acid and glutaconic acid. Clinical features include the sudden onset of encephalopathy, hypotonia and macrocephaly usually before age 18months. Here we report five cases of GA-I confirmed with mutation analysis. GCDH gene mutations were identified in all five probands with GA-I. Three of them had compound heterozygous mutations and two had homozygous mutations. Mutations of two alleles (c.334G>T and IVS11-11A>G) were novel and both of them were confirmed to be splice site mutations by reverse transcription PCR.

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.