Roberto Mendoza-londono - Academia.edu (original) (raw)

Papers by Roberto Mendoza-londono

Research paper thumbnail of COG5 variants lead to complex early onset retinal degeneration, upregulation of PERK and DNA damage

Scientific Reports, 2020

Leber congenital amaurosis (LCA), a form of autosomal recessive severe early-onset retinal degene... more Leber congenital amaurosis (LCA), a form of autosomal recessive severe early-onset retinal degeneration, is an important cause of childhood blindness. This may be associated with systemic features or not. Here we identified COG5 compound-heterozygous variants in patients affected with a complex LCA phenotype associated with microcephaly and skeletal dysplasia. COG5 is a component of the COG complex, which facilitates retrograde Golgi trafficking; if disrupted this can result in protein misfolding. To date, variants in COG5 have been associated with a distinct congenital disorder of glycosylation (type IIi) and with a variant of Friedreich’s ataxia. We show that COG5 variants can also result in fragmentation of the Golgi apparatus and upregulation of the UPR modulator, PKR-like endoplasmic reticulum kinase (PERK). In addition, upregulation of PERK induces DNA damage in cultured cells and in murine retina. This study identifies a novel role for COG5 in maintaining ER protein homeostas...

Research paper thumbnail of Severe Neonatal Cholestasis as an Early Presentation of McCune- Albright Syndrome

Journal of Clinical Research in Pediatric Endocrinology, 2019

McCune-Albright syndrome (MAS) is a rare genetic disorder characterized by café-au-lait macules, ... more McCune-Albright syndrome (MAS) is a rare genetic disorder characterized by café-au-lait macules, polyostotic fibrous dysplasia and multiple endocrinopathies. Liver involvement, although described, is a rare complication. We review the case of a child with MAS whose initial presentation was characterized by severe neonatal cholestasis. The case demonstrates a severe phenotype of persistent cholestasis in MAS requiring liver transplantation. This phenotype has been previously considered to be a more benign feature. This case highlights the importance of consideration of MAS as an uncommon but important cause of neonatal cholestasis. Early diagnosis may allow for prompt recognition and treatment of other endocrinopathies.

Research paper thumbnail of Periodic reanalysis of whole-genome sequencing data enhances the diagnostic advantage over standard clinical genetic testing

European journal of human genetics : EJHG, Jan 16, 2018

Whole-genome sequencing (WGS) as a first-tier diagnostic test could transform medical genetic ass... more Whole-genome sequencing (WGS) as a first-tier diagnostic test could transform medical genetic assessments, but there are limited data regarding its clinical use. We previously showed that WGS could feasibly be deployed as a single molecular test capable of a higher diagnostic rate than current practices, in a prospectively recruited cohort of 100 children meeting criteria for chromosomal microarray analysis. In this study, we report on the added diagnostic yield with re-annotation and reanalysis of these WGS data ~2 years later. Explanatory variants have been discovered in seven (10.9%) of 64 previously undiagnosed cases, in emerging disease genes like HMGA2. No new genetic diagnoses were made by any other method in the interval period as part of ongoing clinical care. The results increase the cumulative diagnostic yield of WGS in the study cohort to 41%. This represents a greater than 5-fold increase over the chromosomal microarrays, and a greater than 3-fold increase over all the ...

Research paper thumbnail of Improved diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test

Genetics in medicine : official journal of the American College of Medical Genetics, Apr 3, 2017

PurposeGenetic testing is an integral diagnostic component of pediatric medicine. Standard of car... more PurposeGenetic testing is an integral diagnostic component of pediatric medicine. Standard of care is often a time-consuming stepwise approach involving chromosomal microarray analysis and targeted gene sequencing panels, which can be costly and inconclusive. Whole-genome sequencing (WGS) provides a comprehensive testing platform that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use.MethodsWe prospectively recruited 103 patients from pediatric non-genetic subspecialty clinics, each with a clinical phenotype suggestive of an underlying genetic disorder, and compared the diagnostic yield and coverage of WGS with those of conventional genetic testing.ResultsWGS identified diagnostic variants in 41% of individuals, representing a significant increase over conventional testing results (24%; P = 0.01). Genes clinically sequenced in the cohort (n = 1,226) were well covered by WGS, with a median exonic coverage of 40 × ±8 ...

Research paper thumbnail of Biallelic mutations in DNAJC21 cause Shwachman-Diamond syndrome

Blood, 2017

There is an Inside Blood Commentary on this article in this issue.

Research paper thumbnail of Mutations in EXTL3 Cause Neuro-immuno-skeletal Dysplasia Syndrome

American journal of human genetics, Feb 2, 2017

EXTL3 regulates the biosynthesis of heparan sulfate (HS), important for both skeletal development... more EXTL3 regulates the biosynthesis of heparan sulfate (HS), important for both skeletal development and hematopoiesis, through the formation of HS proteoglycans (HSPGs). By whole-exome sequencing, we identified homozygous missense mutations c.1382C>T, c.1537C>T, c.1970A>G, and c.2008T>G in EXTL3 in nine affected individuals from five unrelated families. Notably, we found the identical homozygous missense mutation c.1382C>T (p.Pro461Leu) in four affected individuals from two unrelated families. Affected individuals presented with variable skeletal abnormalities and neurodevelopmental defects. Severe combined immunodeficiency (SCID) with a complete absence of T cells was observed in three families. EXTL3 was most abundant in hematopoietic stem cells and early progenitor T cells, which is in line with a SCID phenotype at the level of early T cell development in the thymus. To provide further support for the hypothesis that mutations in EXTL3 cause a neuro-immuno-skeletal d...

Research paper thumbnail of Whole-genome sequencing expands diagnostic utility and improves clinical management in paediatric medicine

npj Genomic Medicine, 2016

The standard of care for first-tier clinical investigation of the aetiology of congenital malform... more The standard of care for first-tier clinical investigation of the aetiology of congenital malformations and neurodevelopmental disorders is chromosome microarray analysis (CMA) for copy-number variations (CNVs), often followed by gene(s)-specific sequencing searching for smaller insertion–deletions (indels) and single-nucleotide variant (SNV) mutations. Whole-genome sequencing (WGS) has the potential to capture all classes of genetic variation in one experiment; however, the diagnostic yield for mutation detection of WGS compared to CMA, and other tests, needs to be established. In a prospective study we utilised WGS and comprehensive medical annotation to assess 100 patients referred to a paediatric genetics service and compared the diagnostic yield versus standard genetic testing. WGS identified genetic variants meeting clinical diagnostic criteria in 34% of cases, representing a fourfold increase in diagnostic rate over CMA (8%; P value=1.42E−05) alone and more than twofold incre...

Research paper thumbnail of Spell Checking Nature: Versatility of CRISPR/Cas9 for Developing Treatments for Inherited Disorders

American journal of human genetics, Jan 9, 2015

Clustered regularly interspaced short palindromic repeat (CRISPR) has arisen as a frontrunner for... more Clustered regularly interspaced short palindromic repeat (CRISPR) has arisen as a frontrunner for efficient genome engineering. However, the potentially broad therapeutic implications are largely unexplored. Here, to investigate the therapeutic potential of CRISPR/Cas9 in a diverse set of genetic disorders, we establish a pipeline that uses readily obtainable cells from affected individuals. We show that an adapted version of CRISPR/Cas9 increases the amount of utrophin, a known disease modifier in Duchenne muscular dystrophy (DMD). Furthermore, we demonstrate preferential elimination of the dominant-negative FGFR3 c.1138G>A allele in fibroblasts of an individual affected by achondroplasia. Using a previously undescribed approach involving single guide RNA, we successfully removed large genome rearrangement in primary cells of an individual with an X chromosome duplication including MECP2. Moreover, removal of a duplication of DMD exons 18-30 in myotubes of an individual affected...

Research paper thumbnail of Mutations Preventing Regulated Exon Skipping in MET Cause Osteofibrous Dysplasia

American journal of human genetics, Jan 3, 2015

The periosteum contributes to bone repair and maintenance of cortical bone mass. In contrast to t... more The periosteum contributes to bone repair and maintenance of cortical bone mass. In contrast to the understanding of bone development within the epiphyseal growth plate, factors that regulate periosteal osteogenesis have not been studied as intensively. Osteofibrous dysplasia (OFD) is a congenital disorder of osteogenesis and is typically sporadic and characterized by radiolucent lesions affecting the cortical bone immediately under the periosteum of the tibia and fibula. We identified germline mutations in MET, encoding a receptor tyrosine kinase, that segregate with an autosomal-dominant form of OFD in three families and a mutation in a fourth affected subject from a simplex family and with bilateral disease. Mutations identified in all families with dominant inheritance and in the one simplex subject with bilateral disease abolished the splice inclusion of exon 14 in MET transcripts, which resulted in a MET receptor (MET(Δ14)) lacking a cytoplasmic juxtamembrane domain. Splice ex...

Research paper thumbnail of Identification of a Recognizable Progressive Skeletal Dysplasia Caused by RSPRY1 Mutations

The American Journal of Human Genetics, 2015

Skeletal dysplasias are highly variable Mendelian phenotypes. Molecular diagnosis of skeletal dys... more Skeletal dysplasias are highly variable Mendelian phenotypes. Molecular diagnosis of skeletal dysplasias is complicated by their extreme clinical and genetic heterogeneity. We describe a clinically recognizable autosomal-recessive disorder in four affected siblings from a consanguineous Saudi family, comprising progressive spondyloepimetaphyseal dysplasia, short stature, facial dysmorphism, short fourth metatarsals, and intellectual disability. Combined autozygome/exome analysis identified a homozygous frameshift mutation in RSPRY1 with resulting nonsense-mediated decay. Using a gene-centric ''matchmaking'' system, we were able to identify a Peruvian simplex case subject whose phenotype is strikingly similar to the original Saudi family and whose exome sequencing had revealed a likely pathogenic homozygous missense variant in the same gene. RSPRY1 encodes a hypothetical RING and SPRY domain-containing protein of unknown physiological function. However, we detect strong RSPRY1 protein localization in murine embryonic osteoblasts and periosteal cells during primary endochondral ossification, consistent with a role in bone development. This study highlights the role of gene-centric matchmaking tools to establish causal links to genes, especially for rare or previously undescribed clinical entities.

Research paper thumbnail of Recessive Osteogenesis Imperfecta Caused by Missense Mutations in SPARC

American journal of human genetics, Jan 28, 2015

Secreted protein, acidic, cysteine-rich (SPARC) is a glycoprotein that binds to collagen type I a... more Secreted protein, acidic, cysteine-rich (SPARC) is a glycoprotein that binds to collagen type I and other proteins in the extracellular matrix. Using whole-exome sequencing to identify the molecular defect in two unrelated girls with severe bone fragility and a clinical diagnosis of osteogenesis imperfecta type IV, we identified two homozygous variants in SPARC (GenBank: NM_003118.3; c.497G>A [p.Arg166His] in individual 1; c.787G>A [p.Glu263Lys] in individual 2). Published modeling and site-directed mutagenesis studies had previously shown that the residues substituted by these mutations form an intramolecular salt bridge in SPARC and are essential for the binding of SPARC to collagen type I. The amount of SPARC secreted by skin fibroblasts was reduced in individual 1 but appeared normal in individual 2. The migration of collagen type I alpha chains produced by these fibroblasts was mildly delayed on SDS-PAGE gel, suggesting some overmodification of collagen during triple heli...

Research paper thumbnail of The Collagenopathies: Review of Clinical Phenotypes and Molecular Correlations

Current Rheumatology Reports, 2013

Genetic defects of collagen formation (the collagenopathies) affect almost every organ system and... more Genetic defects of collagen formation (the collagenopathies) affect almost every organ system and tissue in the body. They can be grouped by clinical phenotype, which usually correlates with the tissue distribution of the affected collagen subtype. Many of these conditions present in childhood; however, milder phenotypes presenting in adulthood are increasingly recognized. Many are difficult to differentiate clinically. Precise diagnosis by means of genetic testing assists in providing prognosis information, family counseling, and individualized treatment. This review provides an overview of the current range of clinical presentations associated with collagen defects, and the molecular mechanisms important to understanding how the results of genetic testing affect medical care.

Research paper thumbnail of Mutations in PLK4, encoding a master regulator of centriole biogenesis, cause microcephaly, growth failure and retinopathy

Nature genetics, 2014

Centrioles are essential for ciliogenesis. However, mutations in centriole biogenesis genes have ... more Centrioles are essential for ciliogenesis. However, mutations in centriole biogenesis genes have been reported in primary microcephaly and Seckel syndrome, disorders without the hallmark clinical features of ciliopathies. Here we identify mutations in the genes encoding PLK4 kinase, a master regulator of centriole duplication, and its substrate TUBGCP6 in individuals with microcephalic primordial dwarfism and additional congenital anomalies, including retinopathy, thereby extending the human phenotypic spectrum associated with centriole dysfunction. Furthermore, we establish that different levels of impaired PLK4 activity result in growth and cilia phenotypes, providing a mechanism by which microcephaly disorders can occur with or without ciliopathic features.

Research paper thumbnail of De novo ACTA2 mutation causes a novel syndrome of multisystemic smooth muscle dysfunction

American Journal of Medical Genetics Part A, 2010

Smooth muscle cells (SMCs) contract to perform many physiological functions, including regulation... more Smooth muscle cells (SMCs) contract to perform many physiological functions, including regulation of blood flow and pressure in arteries, contraction of the pupils, peristalsis of the gut and voiding of the bladder. SMC lineage in these organs is characterized by cellular expression of the SMC isoform of α-actin, encoded by the ACTA2 gene. We report here on a unique and de novo mutation in ACTA2, R179H, that causes a syndrome characterized by dysfunction of SMCs throughout the body, leading to aortic and cerebrovascular disease, fixed dilated pupils, hypotonic bladder, malrotation and hypoperistalsis of the gut and pulmonary hypertension.

Research paper thumbnail of Spondyloepiphyseal dysplasia Maroteaux type: Report of three patients from two families and exclusion of type II collagen defects

American Journal of Medical Genetics, 2003

Spondyloepiphyseal dysplasia (SED) Maroteaux type is an autosomal dominant skeletal dysplasia, ch... more Spondyloepiphyseal dysplasia (SED) Maroteaux type is an autosomal dominant skeletal dysplasia, characterized by spondylar dysplasia, mild epiphyseal dysplasia of the large joints, and type E-like brachydactyly. These manifestations overlap with those of spondyloperipheral dysplasia (SPD), in which a sporadic case with a mutation of COL2A1 has been reported. We report on three patients (an affected woman and her son and a sporadic case of an affected man) with SED Maroteaux type. The affected adults were severely short along with stubby hands and feet, and one developed myelopathy as a result of thoracolumbar gibbus. The affected child was mildly short at birth, and developed brachydactyly in early childhood. The radiological hallmarks of these patients included severe platyspondyly with squareshaped vertebral bodies, iliac hypoplasia, epiphyseal hypoplasia of the large joints, and strikingly short metacarpals and phalanges. These radiological findings appeared already apparent in early childhood. SED Maroteaux type was radiologically discriminative from SPD. Brachydactyly was much severe in the former than in the latter, and spondylar dysplasia manifestation was different between both disorders. Mutation screen by polymerase chain reaction-direct sequencing for all exons and their flanking regions of COL2A1 did not reveal any mutations in the three patients.

Research paper thumbnail of Mutations in B3GALT6, which Encodes a Glycosaminoglycan Linker Region Enzyme, Cause a Spectrum of Skeletal and Connective Tissue Disorders

The American Journal of Human Genetics, 2013

Proteoglycans (PGs) are a major component of the extracellular matrix in many tissues and functio... more Proteoglycans (PGs) are a major component of the extracellular matrix in many tissues and function as structural and regulatory molecules. PGs are composed of core proteins and glycosaminoglycan (GAG) side chains. The biosynthesis of GAGs starts with the linker region that consists of four sugar residues and is followed by repeating disaccharide units. By exome sequencing, we found that B3GALT6 encoding an enzyme involved in the biosynthesis of the GAG linker region is responsible for a severe skeletal dysplasia, spondyloepimetaphyseal dysplasia with joint laxity type 1 (SEMD-JL1). B3GALT6 loss-of-function mutations were found in individuals with SEMD-JL1 from seven families. In a subsequent candidate gene study based on the phenotypic similarity, we found that B3GALT6 is also responsible for a connective tissue disease, Ehlers-Danlos syndrome (progeroid form). Recessive loss-of-function mutations in B3GALT6 result in a spectrum of disorders affecting a broad range of skeletal and connective tissues characterized by lax skin, muscle hypotonia, joint dislocation, and spinal deformity. The pleiotropic phenotypes of the disorders indicate that B3GALT6 plays a critical role in a wide range of biological processes in various tissues, including skin, bone, cartilage, tendon, and ligament.

Research paper thumbnail of The Ehlers-Danlos syndromes, rare types

American Journal of Medical Genetics Part C: Seminars in Medical Genetics, 2017

Her group is dedicated to the pathobiology of connective tissue disorders, in particular the Ehle... more Her group is dedicated to the pathobiology of connective tissue disorders, in particular the Ehlers-Danlos syndromes, and specializes on the molecular and structural basis of glycosaminoglycan synthesis enzyme defects.

Research paper thumbnail of The Collagenopathies: Review of Clinical Phenotypes and Molecular Correlations

Current Rheumatology Reports, 2013

Genetic defects of collagen formation (the collagenopathies) affect almost every organ system and... more Genetic defects of collagen formation (the collagenopathies) affect almost every organ system and tissue in the body. They can be grouped by clinical phenotype, which usually correlates with the tissue distribution of the affected collagen subtype. Many of these conditions present in childhood; however, milder phenotypes presenting in adulthood are increasingly recognized. Many are difficult to differentiate clinically. Precise diagnosis by means of genetic testing assists in providing prognosis information, family counseling, and individualized treatment. This review provides an overview of the current range of clinical presentations associated with collagen defects, and the molecular mechanisms important to understanding how the results of genetic testing affect medical care.

Research paper thumbnail of COG5 variants lead to complex early onset retinal degeneration, upregulation of PERK and DNA damage

Scientific Reports, 2020

Leber congenital amaurosis (LCA), a form of autosomal recessive severe early-onset retinal degene... more Leber congenital amaurosis (LCA), a form of autosomal recessive severe early-onset retinal degeneration, is an important cause of childhood blindness. This may be associated with systemic features or not. Here we identified COG5 compound-heterozygous variants in patients affected with a complex LCA phenotype associated with microcephaly and skeletal dysplasia. COG5 is a component of the COG complex, which facilitates retrograde Golgi trafficking; if disrupted this can result in protein misfolding. To date, variants in COG5 have been associated with a distinct congenital disorder of glycosylation (type IIi) and with a variant of Friedreich’s ataxia. We show that COG5 variants can also result in fragmentation of the Golgi apparatus and upregulation of the UPR modulator, PKR-like endoplasmic reticulum kinase (PERK). In addition, upregulation of PERK induces DNA damage in cultured cells and in murine retina. This study identifies a novel role for COG5 in maintaining ER protein homeostas...

Research paper thumbnail of Severe Neonatal Cholestasis as an Early Presentation of McCune- Albright Syndrome

Journal of Clinical Research in Pediatric Endocrinology, 2019

McCune-Albright syndrome (MAS) is a rare genetic disorder characterized by café-au-lait macules, ... more McCune-Albright syndrome (MAS) is a rare genetic disorder characterized by café-au-lait macules, polyostotic fibrous dysplasia and multiple endocrinopathies. Liver involvement, although described, is a rare complication. We review the case of a child with MAS whose initial presentation was characterized by severe neonatal cholestasis. The case demonstrates a severe phenotype of persistent cholestasis in MAS requiring liver transplantation. This phenotype has been previously considered to be a more benign feature. This case highlights the importance of consideration of MAS as an uncommon but important cause of neonatal cholestasis. Early diagnosis may allow for prompt recognition and treatment of other endocrinopathies.

Research paper thumbnail of Periodic reanalysis of whole-genome sequencing data enhances the diagnostic advantage over standard clinical genetic testing

European journal of human genetics : EJHG, Jan 16, 2018

Whole-genome sequencing (WGS) as a first-tier diagnostic test could transform medical genetic ass... more Whole-genome sequencing (WGS) as a first-tier diagnostic test could transform medical genetic assessments, but there are limited data regarding its clinical use. We previously showed that WGS could feasibly be deployed as a single molecular test capable of a higher diagnostic rate than current practices, in a prospectively recruited cohort of 100 children meeting criteria for chromosomal microarray analysis. In this study, we report on the added diagnostic yield with re-annotation and reanalysis of these WGS data ~2 years later. Explanatory variants have been discovered in seven (10.9%) of 64 previously undiagnosed cases, in emerging disease genes like HMGA2. No new genetic diagnoses were made by any other method in the interval period as part of ongoing clinical care. The results increase the cumulative diagnostic yield of WGS in the study cohort to 41%. This represents a greater than 5-fold increase over the chromosomal microarrays, and a greater than 3-fold increase over all the ...

Research paper thumbnail of Improved diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test

Genetics in medicine : official journal of the American College of Medical Genetics, Apr 3, 2017

PurposeGenetic testing is an integral diagnostic component of pediatric medicine. Standard of car... more PurposeGenetic testing is an integral diagnostic component of pediatric medicine. Standard of care is often a time-consuming stepwise approach involving chromosomal microarray analysis and targeted gene sequencing panels, which can be costly and inconclusive. Whole-genome sequencing (WGS) provides a comprehensive testing platform that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use.MethodsWe prospectively recruited 103 patients from pediatric non-genetic subspecialty clinics, each with a clinical phenotype suggestive of an underlying genetic disorder, and compared the diagnostic yield and coverage of WGS with those of conventional genetic testing.ResultsWGS identified diagnostic variants in 41% of individuals, representing a significant increase over conventional testing results (24%; P = 0.01). Genes clinically sequenced in the cohort (n = 1,226) were well covered by WGS, with a median exonic coverage of 40 × ±8 ...

Research paper thumbnail of Biallelic mutations in DNAJC21 cause Shwachman-Diamond syndrome

Blood, 2017

There is an Inside Blood Commentary on this article in this issue.

Research paper thumbnail of Mutations in EXTL3 Cause Neuro-immuno-skeletal Dysplasia Syndrome

American journal of human genetics, Feb 2, 2017

EXTL3 regulates the biosynthesis of heparan sulfate (HS), important for both skeletal development... more EXTL3 regulates the biosynthesis of heparan sulfate (HS), important for both skeletal development and hematopoiesis, through the formation of HS proteoglycans (HSPGs). By whole-exome sequencing, we identified homozygous missense mutations c.1382C>T, c.1537C>T, c.1970A>G, and c.2008T>G in EXTL3 in nine affected individuals from five unrelated families. Notably, we found the identical homozygous missense mutation c.1382C>T (p.Pro461Leu) in four affected individuals from two unrelated families. Affected individuals presented with variable skeletal abnormalities and neurodevelopmental defects. Severe combined immunodeficiency (SCID) with a complete absence of T cells was observed in three families. EXTL3 was most abundant in hematopoietic stem cells and early progenitor T cells, which is in line with a SCID phenotype at the level of early T cell development in the thymus. To provide further support for the hypothesis that mutations in EXTL3 cause a neuro-immuno-skeletal d...

Research paper thumbnail of Whole-genome sequencing expands diagnostic utility and improves clinical management in paediatric medicine

npj Genomic Medicine, 2016

The standard of care for first-tier clinical investigation of the aetiology of congenital malform... more The standard of care for first-tier clinical investigation of the aetiology of congenital malformations and neurodevelopmental disorders is chromosome microarray analysis (CMA) for copy-number variations (CNVs), often followed by gene(s)-specific sequencing searching for smaller insertion–deletions (indels) and single-nucleotide variant (SNV) mutations. Whole-genome sequencing (WGS) has the potential to capture all classes of genetic variation in one experiment; however, the diagnostic yield for mutation detection of WGS compared to CMA, and other tests, needs to be established. In a prospective study we utilised WGS and comprehensive medical annotation to assess 100 patients referred to a paediatric genetics service and compared the diagnostic yield versus standard genetic testing. WGS identified genetic variants meeting clinical diagnostic criteria in 34% of cases, representing a fourfold increase in diagnostic rate over CMA (8%; P value=1.42E−05) alone and more than twofold incre...

Research paper thumbnail of Spell Checking Nature: Versatility of CRISPR/Cas9 for Developing Treatments for Inherited Disorders

American journal of human genetics, Jan 9, 2015

Clustered regularly interspaced short palindromic repeat (CRISPR) has arisen as a frontrunner for... more Clustered regularly interspaced short palindromic repeat (CRISPR) has arisen as a frontrunner for efficient genome engineering. However, the potentially broad therapeutic implications are largely unexplored. Here, to investigate the therapeutic potential of CRISPR/Cas9 in a diverse set of genetic disorders, we establish a pipeline that uses readily obtainable cells from affected individuals. We show that an adapted version of CRISPR/Cas9 increases the amount of utrophin, a known disease modifier in Duchenne muscular dystrophy (DMD). Furthermore, we demonstrate preferential elimination of the dominant-negative FGFR3 c.1138G>A allele in fibroblasts of an individual affected by achondroplasia. Using a previously undescribed approach involving single guide RNA, we successfully removed large genome rearrangement in primary cells of an individual with an X chromosome duplication including MECP2. Moreover, removal of a duplication of DMD exons 18-30 in myotubes of an individual affected...

Research paper thumbnail of Mutations Preventing Regulated Exon Skipping in MET Cause Osteofibrous Dysplasia

American journal of human genetics, Jan 3, 2015

The periosteum contributes to bone repair and maintenance of cortical bone mass. In contrast to t... more The periosteum contributes to bone repair and maintenance of cortical bone mass. In contrast to the understanding of bone development within the epiphyseal growth plate, factors that regulate periosteal osteogenesis have not been studied as intensively. Osteofibrous dysplasia (OFD) is a congenital disorder of osteogenesis and is typically sporadic and characterized by radiolucent lesions affecting the cortical bone immediately under the periosteum of the tibia and fibula. We identified germline mutations in MET, encoding a receptor tyrosine kinase, that segregate with an autosomal-dominant form of OFD in three families and a mutation in a fourth affected subject from a simplex family and with bilateral disease. Mutations identified in all families with dominant inheritance and in the one simplex subject with bilateral disease abolished the splice inclusion of exon 14 in MET transcripts, which resulted in a MET receptor (MET(Δ14)) lacking a cytoplasmic juxtamembrane domain. Splice ex...

Research paper thumbnail of Identification of a Recognizable Progressive Skeletal Dysplasia Caused by RSPRY1 Mutations

The American Journal of Human Genetics, 2015

Skeletal dysplasias are highly variable Mendelian phenotypes. Molecular diagnosis of skeletal dys... more Skeletal dysplasias are highly variable Mendelian phenotypes. Molecular diagnosis of skeletal dysplasias is complicated by their extreme clinical and genetic heterogeneity. We describe a clinically recognizable autosomal-recessive disorder in four affected siblings from a consanguineous Saudi family, comprising progressive spondyloepimetaphyseal dysplasia, short stature, facial dysmorphism, short fourth metatarsals, and intellectual disability. Combined autozygome/exome analysis identified a homozygous frameshift mutation in RSPRY1 with resulting nonsense-mediated decay. Using a gene-centric ''matchmaking'' system, we were able to identify a Peruvian simplex case subject whose phenotype is strikingly similar to the original Saudi family and whose exome sequencing had revealed a likely pathogenic homozygous missense variant in the same gene. RSPRY1 encodes a hypothetical RING and SPRY domain-containing protein of unknown physiological function. However, we detect strong RSPRY1 protein localization in murine embryonic osteoblasts and periosteal cells during primary endochondral ossification, consistent with a role in bone development. This study highlights the role of gene-centric matchmaking tools to establish causal links to genes, especially for rare or previously undescribed clinical entities.

Research paper thumbnail of Recessive Osteogenesis Imperfecta Caused by Missense Mutations in SPARC

American journal of human genetics, Jan 28, 2015

Secreted protein, acidic, cysteine-rich (SPARC) is a glycoprotein that binds to collagen type I a... more Secreted protein, acidic, cysteine-rich (SPARC) is a glycoprotein that binds to collagen type I and other proteins in the extracellular matrix. Using whole-exome sequencing to identify the molecular defect in two unrelated girls with severe bone fragility and a clinical diagnosis of osteogenesis imperfecta type IV, we identified two homozygous variants in SPARC (GenBank: NM_003118.3; c.497G>A [p.Arg166His] in individual 1; c.787G>A [p.Glu263Lys] in individual 2). Published modeling and site-directed mutagenesis studies had previously shown that the residues substituted by these mutations form an intramolecular salt bridge in SPARC and are essential for the binding of SPARC to collagen type I. The amount of SPARC secreted by skin fibroblasts was reduced in individual 1 but appeared normal in individual 2. The migration of collagen type I alpha chains produced by these fibroblasts was mildly delayed on SDS-PAGE gel, suggesting some overmodification of collagen during triple heli...

Research paper thumbnail of The Collagenopathies: Review of Clinical Phenotypes and Molecular Correlations

Current Rheumatology Reports, 2013

Genetic defects of collagen formation (the collagenopathies) affect almost every organ system and... more Genetic defects of collagen formation (the collagenopathies) affect almost every organ system and tissue in the body. They can be grouped by clinical phenotype, which usually correlates with the tissue distribution of the affected collagen subtype. Many of these conditions present in childhood; however, milder phenotypes presenting in adulthood are increasingly recognized. Many are difficult to differentiate clinically. Precise diagnosis by means of genetic testing assists in providing prognosis information, family counseling, and individualized treatment. This review provides an overview of the current range of clinical presentations associated with collagen defects, and the molecular mechanisms important to understanding how the results of genetic testing affect medical care.

Research paper thumbnail of Mutations in PLK4, encoding a master regulator of centriole biogenesis, cause microcephaly, growth failure and retinopathy

Nature genetics, 2014

Centrioles are essential for ciliogenesis. However, mutations in centriole biogenesis genes have ... more Centrioles are essential for ciliogenesis. However, mutations in centriole biogenesis genes have been reported in primary microcephaly and Seckel syndrome, disorders without the hallmark clinical features of ciliopathies. Here we identify mutations in the genes encoding PLK4 kinase, a master regulator of centriole duplication, and its substrate TUBGCP6 in individuals with microcephalic primordial dwarfism and additional congenital anomalies, including retinopathy, thereby extending the human phenotypic spectrum associated with centriole dysfunction. Furthermore, we establish that different levels of impaired PLK4 activity result in growth and cilia phenotypes, providing a mechanism by which microcephaly disorders can occur with or without ciliopathic features.

Research paper thumbnail of De novo ACTA2 mutation causes a novel syndrome of multisystemic smooth muscle dysfunction

American Journal of Medical Genetics Part A, 2010

Smooth muscle cells (SMCs) contract to perform many physiological functions, including regulation... more Smooth muscle cells (SMCs) contract to perform many physiological functions, including regulation of blood flow and pressure in arteries, contraction of the pupils, peristalsis of the gut and voiding of the bladder. SMC lineage in these organs is characterized by cellular expression of the SMC isoform of α-actin, encoded by the ACTA2 gene. We report here on a unique and de novo mutation in ACTA2, R179H, that causes a syndrome characterized by dysfunction of SMCs throughout the body, leading to aortic and cerebrovascular disease, fixed dilated pupils, hypotonic bladder, malrotation and hypoperistalsis of the gut and pulmonary hypertension.

Research paper thumbnail of Spondyloepiphyseal dysplasia Maroteaux type: Report of three patients from two families and exclusion of type II collagen defects

American Journal of Medical Genetics, 2003

Spondyloepiphyseal dysplasia (SED) Maroteaux type is an autosomal dominant skeletal dysplasia, ch... more Spondyloepiphyseal dysplasia (SED) Maroteaux type is an autosomal dominant skeletal dysplasia, characterized by spondylar dysplasia, mild epiphyseal dysplasia of the large joints, and type E-like brachydactyly. These manifestations overlap with those of spondyloperipheral dysplasia (SPD), in which a sporadic case with a mutation of COL2A1 has been reported. We report on three patients (an affected woman and her son and a sporadic case of an affected man) with SED Maroteaux type. The affected adults were severely short along with stubby hands and feet, and one developed myelopathy as a result of thoracolumbar gibbus. The affected child was mildly short at birth, and developed brachydactyly in early childhood. The radiological hallmarks of these patients included severe platyspondyly with squareshaped vertebral bodies, iliac hypoplasia, epiphyseal hypoplasia of the large joints, and strikingly short metacarpals and phalanges. These radiological findings appeared already apparent in early childhood. SED Maroteaux type was radiologically discriminative from SPD. Brachydactyly was much severe in the former than in the latter, and spondylar dysplasia manifestation was different between both disorders. Mutation screen by polymerase chain reaction-direct sequencing for all exons and their flanking regions of COL2A1 did not reveal any mutations in the three patients.

Research paper thumbnail of Mutations in B3GALT6, which Encodes a Glycosaminoglycan Linker Region Enzyme, Cause a Spectrum of Skeletal and Connective Tissue Disorders

The American Journal of Human Genetics, 2013

Proteoglycans (PGs) are a major component of the extracellular matrix in many tissues and functio... more Proteoglycans (PGs) are a major component of the extracellular matrix in many tissues and function as structural and regulatory molecules. PGs are composed of core proteins and glycosaminoglycan (GAG) side chains. The biosynthesis of GAGs starts with the linker region that consists of four sugar residues and is followed by repeating disaccharide units. By exome sequencing, we found that B3GALT6 encoding an enzyme involved in the biosynthesis of the GAG linker region is responsible for a severe skeletal dysplasia, spondyloepimetaphyseal dysplasia with joint laxity type 1 (SEMD-JL1). B3GALT6 loss-of-function mutations were found in individuals with SEMD-JL1 from seven families. In a subsequent candidate gene study based on the phenotypic similarity, we found that B3GALT6 is also responsible for a connective tissue disease, Ehlers-Danlos syndrome (progeroid form). Recessive loss-of-function mutations in B3GALT6 result in a spectrum of disorders affecting a broad range of skeletal and connective tissues characterized by lax skin, muscle hypotonia, joint dislocation, and spinal deformity. The pleiotropic phenotypes of the disorders indicate that B3GALT6 plays a critical role in a wide range of biological processes in various tissues, including skin, bone, cartilage, tendon, and ligament.

Research paper thumbnail of The Ehlers-Danlos syndromes, rare types

American Journal of Medical Genetics Part C: Seminars in Medical Genetics, 2017

Her group is dedicated to the pathobiology of connective tissue disorders, in particular the Ehle... more Her group is dedicated to the pathobiology of connective tissue disorders, in particular the Ehlers-Danlos syndromes, and specializes on the molecular and structural basis of glycosaminoglycan synthesis enzyme defects.

Research paper thumbnail of The Collagenopathies: Review of Clinical Phenotypes and Molecular Correlations

Current Rheumatology Reports, 2013

Genetic defects of collagen formation (the collagenopathies) affect almost every organ system and... more Genetic defects of collagen formation (the collagenopathies) affect almost every organ system and tissue in the body. They can be grouped by clinical phenotype, which usually correlates with the tissue distribution of the affected collagen subtype. Many of these conditions present in childhood; however, milder phenotypes presenting in adulthood are increasingly recognized. Many are difficult to differentiate clinically. Precise diagnosis by means of genetic testing assists in providing prognosis information, family counseling, and individualized treatment. This review provides an overview of the current range of clinical presentations associated with collagen defects, and the molecular mechanisms important to understanding how the results of genetic testing affect medical care.