Publications by authors named "Richard Lao"

10 Publications

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Three patients with homozygous familial hypercholesterolemia: Genomic sequencing and kindred analysis.

Mol Genet Genomic Med 2019 12 16;7(12):e1007. Epub 2019 Oct 16.

Cardiovascular Research Institute, University of California, San Francisco, CA, USA.

Background: Homozygous Familial Hypercholesterolemia (HoFH) is an inherited recessive condition associated with extremely high levels of low-density lipoprotein (LDL) cholesterol in affected individuals. It is usually caused by homozygous or compound heterozygous functional mutations in the LDL receptor (LDLR). A number of mutations causing FH have been reported in literature and such genetic heterogeneity presents great challenges for disease diagnosis.

Objective: We aim to determine the likely genetic defects responsible for three cases of pediatric HoFH in two kindreds.

Methods: We applied whole exome sequencing (WES) on the two probands to determine the likely functional variants among candidate FH genes. We additionally applied 10x Genomics (10xG) Linked-Reads whole genome sequencing (WGS) on one of the kindreds to identify potentially deleterious structural variants (SVs) underlying HoFH. A PCR-based screening assay was also established to detect the LDLR structural variant in a cohort of 641 patients with elevated LDL.

Results: In the Caucasian kindred, the FH homozygosity can be attributed to two compound heterozygous LDLR damaging variants, an exon 12 p.G592E missense mutation and a novel 3kb exon 1 deletion. By analyzing the 10xG phased data, we ascertained that this deletion allele was most likely to have originated from a Russian ancestor. In the Mexican kindred, the strikingly elevated LDL cholesterol level can be attributed to a homozygous frameshift LDLR variant p.E113fs.

Conclusions: While the application of WES can provide a cost-effective way of identifying the genetic causes of FH, it often lacks sensitivity for detecting structural variants. Our finding of the LDLR exon 1 deletion highlights the broader utility of Linked-Read WGS in detecting SVs in the clinical setting, especially when HoFH patients remain undiagnosed after WES.
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http://dx.doi.org/10.1002/mgg3.1007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900368PMC
December 2019

A recurrent, non-penetrant sequence variant, p.Arg266Cys in Growth/Differentiation Factor 3 () in a female with unilateral anophthalmia and skeletal anomalies.

Am J Ophthalmol Case Rep 2017 Sep 21;7:102-106. Epub 2017 Jun 21.

Dept. of Pediatrics, Division of Genetics, University of California San Francisco, San Francisco, CA, USA.

Purpose: The genetic causes of anophthalmia, microphthalmia and coloboma remain poorly understood. Missense mutations in Growth/Differentiation Factor 3 () gene have previously been reported in patients with microphthalmia, iridial and retinal colobomas, Klippel-Feil anomaly with vertebral fusion, scoliosis, rudimentary 12th ribs and an anomalous right temporal bone. We used whole exome sequencing with a trio approach to study a female with unilateral anophthalmia, kyphoscoliosis and additional skeletal anomalies.

Observations: Exome sequencing revealed that the proposita was heterozygous for c.796C > T, predicting p.Arg266Cys, in . Sanger sequencing confirmed the mutation and showed that the unaffected mother was heterozygous for the same missense substitution.

Conclusions And Importance: Although transfection studies with the p.Arg266Cys mutation have shown that this amino acid substitution is likely to impair function, non-penetrance for the ocular defects was apparent in this family and has been observed in other families with sequence variants in . We conclude p.Arg266Cys and other mutations can be non-penetrant, making pathogenicity more difficult to establish when sequence variants in this gene are present in patients with structural eye defects.
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http://dx.doi.org/10.1016/j.ajoc.2017.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5722175PMC
September 2017

Whole exome and whole genome sequencing with dried blood spot DNA without whole genome amplification.

Hum Mutat 2018 01 6;39(1):167-171. Epub 2017 Nov 6.

Cardiovascular Research Institute, University of California, San Francisco, California.

Newborn screening (NBS) for rare conditions is performed in all 50 states in the USA. We have partnered with the California Department of Public Health Genetic Disease Laboratory to determine whether sufficient DNA can be extracted from archived dried blood spots (DBS) for next-generation sequencing in the hopes that next-generation sequencing can play a role in NBS. We optimized the DNA extraction and sequencing library preparation protocols for residual infant DBS archived over 20 years ago and successfully obtained acceptable whole exome and whole genome sequencing data. This sequencing study using DBS DNA without whole genome amplification prior to sequencing library preparation provides evidence that properly stored residual newborn DBS are a satisfactory source of DNA for genetic studies.
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http://dx.doi.org/10.1002/humu.23356DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5738671PMC
January 2018

Genetic analysis of consanguineous families presenting with congenital ocular defects.

Exp Eye Res 2016 05 16;146:163-171. Epub 2016 Mar 16.

Department of Pediatrics, University of California, San Francisco, CA, USA. Electronic address:

Anophthalmia and microphthalmia (A/M) are a group of rare developmental disorders that affect the size of the ocular globe. A/M may present as the sole clinical feature, but are also frequently found in a variety of syndromes. A/M is genetically heterogeneous and can be caused by chromosomal aberrations, copy number variations and single gene mutations. To date, A/M has been caused by mutations in at least 20 genes that show different modes of inheritance. In this study, we enrolled eight consanguineous families with A/M, including seven from Pakistan and one from India. Sanger and exome sequencing of DNA samples from these families identified three novel mutations including two mutations in the Aldehyde Dehydrogenase 1 Family Member A3 (ALDH1A3) gene, [c.1310_1311delAT; p.(Tyr437Trpfs*44) and c.964G > A; p.(Val322Met)] and a single missense mutation in Forkhead Box E3 (FOXE3) gene, [c.289A > G p.(Ile97Val)]. Additionally two previously reported mutations were identified in FOXE3 and in Visual System Homeobox 2 (VSX2). This is the first comprehensive study on families with A/M from the Indian subcontinent which provides further evidence for the involvement of known genes with novel and recurrent mutations.
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http://dx.doi.org/10.1016/j.exer.2016.03.014DOI Listing
May 2016

Genotyping Informatics and Quality Control for 100,000 Subjects in the Genetic Epidemiology Research on Adult Health and Aging (GERA) Cohort.

Genetics 2015 Aug 19;200(4):1051-60. Epub 2015 Jun 19.

Kaiser Permanente Northern California Division of Research, Oakland, California 94612.

The Kaiser Permanente (KP) Research Program on Genes, Environment and Health (RPGEH), in collaboration with the University of California-San Francisco, undertook genome-wide genotyping of >100,000 subjects that constitute the Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort. The project, which generated >70 billion genotypes, represents the first large-scale use of the Affymetrix Axiom Genotyping Solution. Because genotyping took place over a short 14-month period, creating a near-real-time analysis pipeline for experimental assay quality control and final optimized analyses was critical. Because of the multi-ethnic nature of the cohort, four different ethnic-specific arrays were employed to enhance genome-wide coverage. All assays were performed on DNA extracted from saliva samples. To improve sample call rates and significantly increase genotype concordance, we partitioned the cohort into disjoint packages of plates with similar assay contexts. Using strict QC criteria, the overall genotyping success rate was 103,067 of 109,837 samples assayed (93.8%), with a range of 92.1-95.4% for the four different arrays. Similarly, the SNP genotyping success rate ranged from 98.1 to 99.4% across the four arrays, the variation depending mostly on how many SNPs were included as single copy vs. double copy on a particular array. The high quality and large scale of genotype data created on this cohort, in conjunction with comprehensive longitudinal data from the KP electronic health records of participants, will enable a broad range of highly powered genome-wide association studies on a diversity of traits and conditions.
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http://dx.doi.org/10.1534/genetics.115.178905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574249PMC
August 2015

DLX4 is associated with orofacial clefting and abnormal jaw development.

Hum Mol Genet 2015 Aug 7;24(15):4340-52. Epub 2015 May 7.

Department of Pediatrics, University of California, San Francisco, San Francisco, CA 94143, USA, Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA,

Cleft lip and/or palate (CL/P) are common structural birth defects in humans. We used exome sequencing to study a patient with bilateral CL/P and identified a single nucleotide deletion in the patient and her similarly affected son—c.546_546delG, predicting p.Gln183Argfs*57 in the Distal-less 4 (DLX4) gene. The sequence variant was absent from databases, predicted to be deleterious and was verified by Sanger sequencing. In mammals, there are three Dlx homeobox clusters with closely located gene pairs (Dlx1/Dlx2, Dlx3/Dlx4, Dlx5/Dlx6). In situ hybridization showed that Dlx4 was expressed in the mesenchyme of the murine palatal shelves at E12.5, prior to palate closure. Wild-type human DLX4, but not mutant DLX4_c.546delG, could activate two murine Dlx conserved regulatory elements, implying that the mutation caused haploinsufficiency. We showed that reduced DLX4 expression after short interfering RNA treatment in a human cell line resulted in significant up-regulation of DLX3, DLX5 and DLX6, with reduced expression of DLX2 and significant up-regulation of BMP4, although the increased BMP4 expression was demonstrated only in HeLa cells. We used antisense morpholino oligonucleotides to target the orthologous Danio rerio gene, dlx4b, and found reduced cranial size and abnormal cartilaginous elements. We sequenced DLX4 in 155 patients with non-syndromic CL/P and CP, but observed no sequence variants. From the published literature, Dlx1/Dlx2 double homozygous null mice and Dlx5 homozygous null mice both have clefts of the secondary palate. This first finding of a DLX4 mutation in a family with CL/P establishes DLX4 as a potential cause of human clefts.
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http://dx.doi.org/10.1093/hmg/ddv167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4492397PMC
August 2015

Novel mutations in PXDN cause microphthalmia and anterior segment dysgenesis.

Eur J Hum Genet 2015 Mar 18;23(3):337-41. Epub 2014 Jun 18.

Department of Pediatrics, Division of Genetics, University of California, San Francisco, CA, USA.

We used exome sequencing to study a non-consanguineous family with two children who had anterior segment dysgenesis, sclerocornea, microphthalmia, hypotonia and developmental delays. Sanger sequencing verified two Peroxidasin (PXDN) mutations in both sibs--a maternally inherited, nonsense mutation, c.1021C>T predicting p.(Arg341*), and a paternally inherited, 23-basepair deletion causing a frameshift and premature protein truncation, c.2375_2397del23, predicting p.(Leu792Hisfs*67). We re-examined exome data from 20 other patients with structural eye defects and identified two additional PXDN mutations in a sporadic male with bilateral microphthalmia, cataracts and anterior segment dysgenesis--a maternally inherited, frameshift mutation, c.1192delT, predicting p.(Tyr398Thrfs*40) and a paternally inherited, missense substitution that was predicted to be deleterious, c.947 A>C, predicting p.(Gln316Pro). Mutations in PXDN were previously reported in three families with congenital cataracts, microcornea, sclerocornea and developmental glaucoma. The gene is expressed in corneal epithelium and is secreted into the extracellular matrix. Defective peroxidasin has been shown to impair sulfilimine bond formation in collagen IV, a constituent of the basement membrane, implying that the eye defects result because of loss of basement membrane integrity in the developing eye. Our finding of a broader phenotype than previously appreciated for PXDN mutations is typical for exome-sequencing studies, which have proven to be highly effective for mutation detection in patients with atypical presentations. We conclude that PXDN sequencing should be considered in microphthalmia with anterior segment dysgenesis.
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http://dx.doi.org/10.1038/ejhg.2014.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4326713PMC
March 2015

Focal facial dermal dysplasia, type IV, is caused by mutations in CYP26C1.

Hum Mol Genet 2013 Feb 16;22(4):696-703. Epub 2012 Nov 16.

Division of Genetics, Department of Pediatrics, University of California, San Francisco, San Francisco, CA 94143-0316, USA.

Focal facial dermal dysplasia (FFDD) Type IV is a rare syndrome characterized by facial lesions resembling aplasia cutis in a preauricular distribution along the line of fusion of the maxillary and mandibular prominences. To identify the causative gene(s), exome sequencing was performed in a family with two affected siblings. Assuming autosomal recessive inheritance, two novel sequence variants were identified in both siblings in CYP26C1-a duplication of seven base pairs, which was maternally inherited, c.844_851dupCCATGCA, predicting p.Glu284fsX128 and a missense mutation, c.1433G>A, predicting p.Arg478His, that was paternally inherited. The duplication predicted a frameshift mutation that led to a premature stop codon and premature chain termination, whereas the missense mutation was not functional based on its in vitro expression in mammalian cells. The FFDD skin lesions arise along the sites of fusion of the maxillary and mandibular prominences early in facial development, and Cyp26c1 was expressed exactly along the fusion line for these facial prominences in the first branchial arch in mice. Sequencing of four additional, unrelated Type IV FFDD patients and eight Type II or III TWIST2-negative FFDD patients revealed that three of the Type IV patients were homozygous for the duplication, whereas none of the Type II or III patients had CYP26C1 mutations. The seven base pairs duplication was present in 0.3% of healthy controls and 0.3% of patients with other birth defects. These findings suggest that the phenotypic manifestations of FFDD Type IV can be non-penetrant or underascertained. Thus, FFDD Type IV results from the loss of function mutations in CYP26C1.
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http://dx.doi.org/10.1093/hmg/dds477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3554199PMC
February 2013

Next generation genome-wide association tool: design and coverage of a high-throughput European-optimized SNP array.

Genomics 2011 Aug 30;98(2):79-89. Epub 2011 Apr 30.

Institute for Human Genetics, University of California, San Francisco 94143-0794, CA, USA.

The success of genome-wide association studies has paralleled the development of efficient genotyping technologies. We describe the development of a next-generation microarray based on the new highly-efficient Affymetrix Axiom genotyping technology that we are using to genotype individuals of European ancestry from the Kaiser Permanente Research Program on Genes, Environment and Health (RPGEH). The array contains 674,517 SNPs, and provides excellent genome-wide as well as gene-based and candidate-SNP coverage. Coverage was calculated using an approach based on imputation and cross validation. Preliminary results for the first 80,301 saliva-derived DNA samples from the RPGEH demonstrate very high quality genotypes, with sample success rates above 94% and over 98% of successful samples having SNP call rates exceeding 98%. At steady state, we have produced 462 million genotypes per week for each Axiom system. The new array provides a valuable addition to the repertoire of tools for large scale genome-wide association studies.
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http://dx.doi.org/10.1016/j.ygeno.2011.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3146553PMC
August 2011
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