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1.  A web-based resource to aid comparative and functional analysis of the insulin and IGF-1 receptor family 
Human mutation  2007;28(7):660-668.
The metazoan receptors for insulin, insulin-like growth factor 1 and other insulin-like molecules are transmembrane tyrosine kinases involved in the regulation of cell size, cell proliferation, development, signalling of nutritional and environmental conditions, and aging. Historically, mutations in the human insulin receptor have been studied because such changes often lead to severe insulin resistance. More recently, amino acid sequence alterations in the insulin receptor-like receptors of Drosophila melanogaster and Caenorhabditis elegans, as well as in the mouse insulin receptor have been the focus of attention: These modifications can have profound effects on growth, body size, metabolism and aging. In order to integrate the many findings on insulin/IGF1 receptor structure and function across species we have created Receptors for Insulin and Insulin-like Molecules (RILM), a curated computer-based resource that displays residue-by residue information on sequence homology, three-dimensional structure, structure/function annotation and documented mutations. The resource includes data obtained from sequence and structure analysis tools, primary database resources, and published reports. The information is integrated via a structure-based multiple sequence alignment of diverse members of the family. RILM was designed to provide easy access to multiple data types that could prove useful in the analysis of the effect of mutations on protein structure and ligand binding within this receptor family. RILM is available at
PMCID: PMC4335190  PMID: 17318838
insulin receptor; IGF1 receptor; DAF-2; dInR; mutation; structure; sequence alignment
2.  Calibration of Multiple In Silico Tools for Predicting Pathogenicity of Mismatch Repair Gene Missense Substitutions 
Human mutation  2012;34(1):255-265.
Classification of rare missense substitutions observed during genetic testing for patient management is a considerable problem in clinical genetics. The Bayesian integrated evaluation of unclassified variants is a solution originally developed for BRCA1/2. Here, we take a step toward an analogous system for the mismatch repair (MMR) genes (MLH1, MSH2, MSH6, and PMS2) that confer colon cancer susceptibility in Lynch syndrome by calibrating in silico tools to estimate prior probabilities of pathogenicity for MMR gene missense substitutions. A qualitative five-class classification system was developed and applied to 143 MMR missense variants. This identified 74 missense substitutions suitable for calibration. These substitutions were scored using six different in silico tools (Align-Grantham Variation Grantham Deviation, multivariate analysis of protein polymorphisms [MAPP], Mut-Pred, PolyPhen-2.1, Sorting Intolerant From Tolerant, and Xvar), using curated MMR multiple sequence alignments where possible. The output from each tool was calibrated by regression against the classifications of the 74 missense substitutions; these calibrated outputs are interpretable as prior probabilities of pathogenicity. MAPP was the most accurate tool and MAPP + PolyPhen-2.1 provided the best-combined model (R2 = 0.62 and area under receiver operating characteristic = 0.93). The MAPP + PolyPhen-2.1 output is sufficiently predictive to feed as a continuous variable into the quantitative Bayesian integrated evaluation for clinical classification of MMR gene missense substitutions.
PMCID: PMC4318556  PMID: 22949387
mismatch repair; in silico; missense substitutions; probability of pathogenicity
3.  [No title available] 
PMCID: PMC3935516  PMID: 24186849
4.  [No title available] 
PMCID: PMC3995136  PMID: 24323938
5.  The ZZ Domain of Dystrophin in DMD: Making Sense of Missense Mutations 
Human mutation  2013;35(2):257-264.
Duchenne muscular dystrophy (DMD) is associated with the loss of dystrophin, which plays an important role in myofiber integrity via interactions with β-dystroglycan and other members of the transmembrane dystrophin-associated protein complex. The ZZ domain, a cysteine-rich zinc-finger domain near the dystrophin C-terminus, is implicated in forming a stable interaction between dystrophin and β-dystroglycan, but the mechanism of pathogenesis of ZZ missense mutations has remained unclear because not all such mutations have been shown to alter β-dystroglycan binding in previous experimental systems. We engineered three ZZ mutations (p.Cys3313Phe, p.Asp3335His, and p.Cys3340Tyr) into a short construct similar to the Dp71 dystrophin isoform for in vitro and in vivo studies and delineated their effect on protein expression, folding properties, and binding partners. Our results demonstrate two distinct pathogenic mechanisms for ZZ missense mutations. The cysteine mutations result in diminished or absent subsarcolemmal expression because of protein instability, likely due to misfolding. In contrast, the aspartic acid mutation disrupts binding with β-dystroglycan despite an almost normal expression at the membrane, confirming a role for the ZZ domain in β-dystroglycan binding but surprisingly demonstrating that such binding is not required for subsarcolemmal localization of dystrophin, even in the absence of actin binding domains.
PMCID: PMC4145872  PMID: 24302611
Duchenne muscular dystrophy; dystrophin; missense mutation; ZZ domain
6.  [No title available] 
PMCID: PMC4292933  PMID: 18566966
7.  Reflecting on Earlier Experiences with Unsolicited Findings: Points to Consider for Next-Generation Sequencing and Informed Consent in Diagnostics 
Human Mutation  2013;34(10):1322-1328.
High-throughput nucleotide sequencing (often referred to as next-generation sequencing; NGS) is increasingly being chosen as a diagnostic tool for cases of expected but unresolved genetic origin. When exploring a higher number of genetic variants, there is a higher chance of detecting unsolicited findings. The consequential increased need for decisions on disclosure of these unsolicited findings poses a challenge for the informed consent procedure. This article discusses the ethical and practical dilemmas encountered when contemplating informed consent for NGS in diagnostics from a multidisciplinary point of view. By exploring recent similar experiences with unsolicited findings in other settings, an attempt is made to describe what can be learned so far for implementing NGS in standard genetic diagnostics. The article concludes with a set of points to consider in order to guide decision-making on the extent of return of results in relation to the mode of informed consent. We hereby aim to provide a sound basis for developing guidelines for optimizing the informed consent procedure.
PMCID: PMC4285964  PMID: 23784691
high-throughput nucleotide sequencing; incidental findings; unsolicited findings; diagnosis; informed consent
8.  DIAMUND: Direct Comparison of Genomes to Detect Mutations 
Human Mutation  2013;35(3):283-288.
DNA sequencing has become a powerful method to discover the genetic basis of disease. Standard, widely used protocols for analysis usually begin by comparing each individual to the human reference genome. When applied to a set of related individuals, this approach reveals millions of differences, most of which are shared among the individuals and unrelated to the disease being investigated. We have developed a novel algorithm for variant detection, one that compares DNA sequences directly to one another, without aligning them to the reference genome. When used to find de novo mutations in exome sequences from family trios, or to compare normal and diseased samples from the same individual, the new method, direct alignment for mutation discovery (DIAMUND), produces a dramatically smaller list of candidate mutations than previous methods, without losing sensitivity to detect the true cause of a genetic disease. We demonstrate our results on several example cases, including two family trios in which it correctly found the disease-causing variant while excluding thousands of harmless variants that standard methods had identified.
PMCID: PMC4031744  PMID: 24375697
variant detection; computational biology; bioinformatics; exome sequencing; sequence alignment
9.  Genetic Variations and Diseases in UniProtKB/Swiss-Prot: The Ins and Outs of Expert Manual Curation 
Human Mutation  2014;35(8):927-935.
During the last few years, next-generation sequencing (NGS) technologies have accelerated the detection of genetic variants resulting in the rapid discovery of new disease-associated genes. However, the wealth of variation data made available by NGS alone is not sufficient to understand the mechanisms underlying disease pathogenesis and manifestation. Multidisciplinary approaches combining sequence and clinical data with prior biological knowledge are needed to unravel the role of genetic variants in human health and disease. In this context, it is crucial that these data are linked, organized, and made readily available through reliable online resources. The Swiss-Prot section of the Universal Protein Knowledgebase (UniProtKB/Swiss-Prot) provides the scientific community with a collection of information on protein functions, interactions, biological pathways, as well as human genetic diseases and variants, all manually reviewed by experts. In this article, we present an overview of the information content of UniProtKB/Swiss-Prot to show how this knowledgebase can support researchers in the elucidation of the mechanisms leading from a molecular defect to a disease phenotype.
PMCID: PMC4107114  PMID: 24848695
UniProtKB/Swiss-Prot; database; manual curation; genetic variants; disease; functional annotation; controlled vocabulary
10.  Three Different Cone Opsin Gene Array Mutational Mechanisms with Genotype–Phenotype Correlation and Functional Investigation of Cone Opsin Variants 
Human Mutation  2014;35(11):1354-1362.
Mutations in the OPN1LW (L-) and OPN1MW (M-)cone opsin genes underlie a spectrum of cone photoreceptor defects from stationary loss of color vision to progressive retinal degeneration. Genotypes of 22 families with a range of cone disorders were grouped into three classes: deletions of the locus control region (LCR); missense mutation (p.Cys203Arg) in an L-/M-hybrid gene; and exon 3 single-nucleotide polymorphism (SNP) interchange haplotypes in an otherwise normal gene array. Moderate-to-high myopia was observed in all mutation categories. Individuals with LCR deletions or p.Cys203Arg mutations were more likely to have nystagmus and poor vision, with disease progression in some p.Cys203Arg patients. Three disease-associated exon 3 SNP haplotypes encoding LIAVA, LVAVA, or MIAVA were identified in our cohort. These patients were less likely to have nystagmus but more likely to show progression, with all patients over the age of 40 years having marked macular abnormalities. Previously, the haplotype LIAVA has been shown to result in exon 3 skipping. Here, we show that haplotypes LVAVA and MIAVA also result in aberrant splicing, with a residual low level of correctly spliced cone opsin. The OPN1LW/OPN1MW:c.532A>G SNP, common to all three disease-associated haplotypes, appears to be principally responsible for this mutational mechanism.
PMCID: PMC4285181  PMID: 25168334
opsin; blue cone monochromacy; splicing; cone dystrophy; OPN1LW; OPN1MW
11.  Structural Genomic Variation as Risk Factor for Idiopathic Recurrent Miscarriage 
Human Mutation  2014;35(8):972-982.
Recurrent miscarriage (RM) is a multifactorial disorder with acknowledged genetic heritability that affects ∼3% of couples aiming at childbirth. As copy number variants (CNVs) have been shown to contribute to reproductive disease susceptibility, we aimed to describe genome-wide profile of CNVs and identify common rearrangements modulating risk to RM. Genome-wide screening of Estonian RM patients and fertile controls identified excessive cumulative burden of CNVs (5.4 and 6.1 Mb per genome) in two RM cases possibly increasing their individual disease risk. Functional profiling of all rearranged genes within RM study group revealed significant enrichment of loci related to innate immunity and immunoregulatory pathways essential for immune tolerance at fetomaternal interface. As a major finding, we report a multicopy duplication (61.6 kb) at 5p13.3 conferring increased maternal risk to RM in Estonia and Denmark (meta-analysis, n = 309/205, odds ratio = 4.82, P = 0.012). Comparison to Estonian population-based cohort (total, n = 1000) confirmed the risk for Estonian female cases (P = 7.9 × 10−4). Datasets of four cohorts from the Database of Genomic Variants (total, n = 5,846 subjects) exhibited similar low duplication prevalence worldwide (0.7%–1.2%) compared to RM cases of this study (6.6%–7.5%). The CNV disrupts PDZD2 and GOLPH3 genes predominantly expressed in placenta and it may represent a novel risk factor for pregnancy complications.
PMCID: PMC4285182  PMID: 24827138
recurrent miscarriage; fetomaternal interface; immune dysfunction; placenta; GOLPH3; PDZD2
12.  Robust Diagnostic Genetic Testing Using Solution Capture Enrichment and a Novel Variant-Filtering Interface 
Human Mutation  2013;35(4):434-441.
Targeted hybridization enrichment prior to next-generation sequencing is a widespread method for characterizing sequence variation in a research setting, and is being adopted by diagnostic laboratories. However, the number of variants identified can overwhelm clinical laboratories with strict time constraints, the final interpretation of likely pathogenicity being a particular bottleneck. To address this, we have developed an approach in which, after automatic variant calling on a standard unix pipeline, subsequent variant filtering is performed interactively, using AgileExomeFilter and AgilePindelFilter (, tools designed for clinical scientists with standard desktop computers. To demonstrate the method's diagnostic efficacy, we tested 128 patients using (1) a targeted capture of 36 cancer-predisposing genes or (2) whole-exome capture for diagnosis of the genetically heterogeneous disorder primary ciliary dyskinesia (PCD). In the cancer cohort, complete concordance with previous diagnostic data was achieved across 793 variant genotypes. A high yield (42%) was also achieved for exome-based PCD diagnosis, underscoring the scalability of our method. Simple adjustments to the variant filtering parameters further allowed the identification of a homozygous truncating mutation in a presumptive new PCD gene, DNAH8. These tools should allow diagnostic laboratories to expand their testing portfolios flexibly, using a standard set of reagents and techniques.
PMCID: PMC4285299  PMID: 24307375
software; exome sequencing; sequence analysis; mutation detection
13.  Exome Sequencing Identifies Potential Risk Variants for Mendelian Disorders at High Prevalence in Qatar 
Human mutation  2013;35(1):105-116.
Exome sequencing of families of related individuals has been highly successful in identifying genetic polymorphisms responsible for Mendelian disorders. Here, we demonstrate the value of the reverse approach, where we use exome sequencing of a sample of unrelated individuals to analyze allele frequencies of known causal mutations for Mendelian diseases. We sequenced the exomes of 100 individuals representing the three major genetic subgroups of the Qatari population (Q1 Bedouin, Q2 Persian-South Asian, Q3 African) and identified 37 variants in 33 genes with effects on 36 clinically significant Mendelian diseases. These include variants not present in 1000 Genomes and variants at high frequency when compared to 1000 Genomes populations. Several of these Mendelian variants were only segregating in one Qatari subpopulation, where the observed subpopulation specificity trends were confirmed in an independent population of 386 Qataris. Pre-marital genetic screening in Qatar tests for only 4 out of the 37, such that this study provides a set of Mendelian disease variants with potential impact on the epidemiological profile of the population that could be incorporated into the testing program if further experimental and clinical characterization confirms high penetrance.
PMCID: PMC3908915  PMID: 24123366
Mendelian; consanguinity; exome sequencing; OMIM; diagnostic biomarkers
14.  Genetic and functional analyses of ZIC3 variants in congenital heart disease 
Human mutation  2014;35(1):66-75.
Mutations in zinc-finger in cerebellum 3 (ZIC3) result in heterotaxy or isolated congenital heart disease (CHD). The majority of reported mutations cluster in zinc-finger domains. We previously demonstrated that many of these lead to aberrant ZIC3 subcellular trafficking. A relative paucity of N- and C-terminal mutations has, however, prevented similar analyses in these regions. Notably, an N-terminal polyalanine expansion was recently identified in a patient with VACTERL, suggesting a potentially distinct function for this domain. Here, we report ZIC3 sequencing results from 440 unrelated patients with heterotaxy and CHD, the largest cohort yet examined. Variants were identified in 5.2% of sporadic male cases. This rate exceeds previous estimates of 1% and has important clinical implications for genetic testing and risk-based counseling. Eight of 11 were novel, including 5 N-terminal variants. Subsequent functional analyses included 4 additional reported but untested variants. Aberrant cytoplasmic localization and decreased luciferase transactivation were observed for all zinc-finger variants, but not for downstream or in-frame upstream variants, including both analyzed polyalanine expansions. Collectively, these results expand the ZIC3 mutational spectrum, support a higher than expected prevalence in sporadic cases, and suggest alternative functions for terminal mutations, highlighting a need for further study of these domains.
PMCID: PMC3946352  PMID: 24123890
ZIC3; left-right patterning; symmetry; heterotaxy; congenital heart disease
15.  High frequency strand slippage mutations in CTCF in MSI-positive endometrial cancers 
Human mutation  2014;35(1):63-65.
Tumors with defective mismatch repair acquire large numbers of strand slippage mutations including frameshifts in coding sequence repeats. We identified a mutational hotspot, p.T204fs, in the insulator-binding protein (CTCF) in MSI-positive endometrial cancers. Although CTCF was described as a significantly mutated gene by the endometrial cancer TCGA, the A7 track variants leading to T204 frameshifts were not reported. Reanalysis of TCGA data using Pindel revealed frequent T204fs mutations, confirming CTCF is an MSI target gene and revealed the same frameshifts in tumors with intact mismatch repair. We show that T204fs transcripts are subject to nonsense-mediated decay and as such, T204fs mutations are unlikely to act as dominant negatives. The spectrum and pattern of mutations observed is consistent with CTCF acting as a haploinsufficient tumor suppressor.
PMCID: PMC3946358  PMID: 24130125
endometrial cancer; MSI; CTCF; strand slippage mutation; tumor suppressor
16.  A homozygous PDE6D mutation in Joubert syndrome impairs targeting of farnesylated INPP5E protein to the primary cilium 
Human mutation  2014;35(1):137-146.
Joubert syndrome (JS) is characterized by a distinctive cerebellar structural defect, namely the « molar tooth sign ». JS is genetically heterogeneous, involving 18 genes identified to date, which are all required for cilia biogenesis and/or function. In a consanguineous family with JS associated with optic nerve coloboma, kidney hypoplasia and polydactyly, combined exome sequencing and mapping identified a homozygous splice site mutation in PDE6D, encoding a prenyl-binding protein. We found that pde6d depletion in zebrafish leads to renal and retinal developmental anomalies and wild-type but not mutant PDE6D is able to rescue this phenotype. Proteomic analysis identified INPP5E, whose mutations also lead to JS or MORM syndromes, as novel prenyl-dependent cargo of PDE6D. Mutant PDE6D shows reduced binding to INPP5E, which fails to localize to primary cilia in patient fibroblasts and tissues. Furthermore, mutant PDE6D is unable to bind to GTP-bound ARL3, which acts as a cargo-release factor for PDE6D-bound INPP5E. Altogether, these results indicate that PDE6D is required for INPP5E ciliary targeting and suggest a broader role for PDE6D in targeting other prenylated proteins to the cilia. This study identifies PDE6D as a novel JS disease gene and provides the first evidence of prenyl-binding dependent trafficking in ciliopathies.
PMCID: PMC3946372  PMID: 24166846
Joubert syndrome; primary cilia; PDE6D; INPP5E; prenylation
17.  The mismatch repair protein MSH2 is rate-limiting for repeat expansion in a Fragile X premutation mouse model 
Human mutation  2014;35(1):129-136.
Fragile X-associated tremor and ataxia syndrome, Fragile X-associated primary ovarian insufficiency and Fragile X syndrome are Repeat Expansion Diseases caused by expansion of a CGG•CCG-repeat microsatellite in the 5′ UTR of the FMR1 gene. To help understand the expansion mechanism responsible for these disorders we have crossed mice containing ~147 CGG•CCG repeats in the endogenous murine Fmr1 gene with mice containing a null mutation in the gene encoding the mismatch repair protein MSH2. MSH2 mutations are associated with elevated levels of generalized microsatellite instability. However, we show here for the first time that in the FX mouse model all maternally and paternally transmitted expansions require Msh2. Even the loss of one Msh2 allele reduced the intergenerational expansion frequency significantly. Msh2 is also required for all somatic expansions and loss of even one functional Msh2 allele reduced the extent of somatic expansion in some organs. Tissues with lower levels of MSH2 were more sensitive to the loss of a single Msh2 allele. This suggests that MSH2 is rate-limiting for expansion in this mouse model and that MSH2 levels may be a key factor that accounts for tissue-specific differences in expansion risk.
PMCID: PMC3951054  PMID: 24130133
Fragile X-related disorders; FXTAS; FXPOI; Triplet repeat expansion; Mismatch repair; MMR; MSH2
18.  Mismatch Repair Analysis of Inherited MSH2 and/or MSH6 Variation Pairs Found in Cancer Patients 
Human mutation  2012;33(8):1294-1301.
Mismatch repair (MMR) malfunction causes the accumulation of mismatches in the genome leading to genomic instability and cancer. The inactivation of an MMR gene (MSH2, MSH6, MLH1, or PMS2) with an inherited mutation causes Lynch syndrome (LS), a dominant susceptibility to cancer. MMR gene variants of uncertain significance (VUS) may be pathogenic mutations, which cause LS, may result in moderately increased cancer risks, or may be harmless polymorphisms. Our study suggests that an inherited MMR VUS individually assessed as proficient may, however, in a pair with another MMR VUS found in the same colorectal cancer (CRC) patient have a concomitant contribution to the MMR deficiency. Here, eight pairs of MMR gene variants found in cancer patients were functionally analyzed in an in vitro MMR assay. Although the other pairs do not suggest a compound deficiency, the MSH2 VUS pair c.380A>G/c.982G>C (p.Asn127Ser/p.Ala328Pro), which nearly halves the repair capability of the wild-type MSH2 protein, is presumed to increase the cancer risk considerably. Moreover, two MSH6 variants, c.1304T>C (p.Leu435Pro) and c.1754T>C (p.Leu585Pro), were shown to be MMR deficient. The role of one of the most frequently reported MMR gene VUS, MSH2 c.380A>G (p.Asn127Ser), is especially interesting because its concomitant defect with another variant could finally explain its recurrent occurrence in CRC patients.
PMCID: PMC4273566  PMID: 22581703
functional analysis; Lynch syndrome; mismatch repair; MSH2; MSH6; VUS
19.  Two Novel Mutations in ABHD12: Expansion of the Mutation Spectrum in PHARC and Assessment of their Functional Effects 
Human mutation  2013;34(12):1672-1678.
PHARC (Polyneuropathy, Hearing loss, Ataxia, Retinitis pigmentosa, and Cataracts) is a recently described autosomal recessive neurodegenerative disease caused by mutations in the α–β–hydrolase domain-containing 12 gene (ABHD12). Only five homozygous ABHD12 mutations have been reported and the pathogenesis of PHARC remains unclear. We evaluated a woman who manifested short stature as well as the typical features of PHARC. Sequence analysis of ABHD12 revealed a novel heterozygous c.1129A>T (p.Lys377X) mutation. Targeted comparative genomic hybridization detected a 59 kb deletion that encompasses exon 1 of ABHD12 and exons 1–4 of an adjacent gene, GINS1, and includes the promoters of both genes. The heterozygous deletion was also carried by the patient’s asymptomatic mother. qRT-PCR demonstrated ~50% decreased expression of ABHD12 RNA in lymphoblastoid cell lines from both individuals. Activity-based protein profiling of serine hydrolases revealed absence of ABHD12 hydrolase activity in the patient and 50% reduction in her mother. This is the first report of compound heterozygosity in PHARC and the first study to describe how a mutation might affect ABHD12 expression and function. The possible involvement of haploinsufficiency for GINS1, a DNA replication complex protein, in the short stature of the patient and her mother requires further studies.
PMCID: PMC3855015  PMID: 24027063
PHARC; ABHD12; GINS1; short stature; endocannabinoid; hydrolase activity
20.  Persistence of Intracellular and Extracellular Changes after Incompletely Suppressing Expression of the R789C (p.R989C) and R992C (p.R1192C) Collagen II Mutants 
Human mutation  2011;32(7):794-805.
Mutations in COL2A1 produce a spectrum of disorders whose hallmark feature is alterations in skeletal development. Attempts to counteract the effects of collagen mutations at the molecular level have been relatively ineffective due to the inability to selectively suppress a mutant allele, and failure to deliver a sufficient number of cells expressing wild-type collagen. Moreover, these approaches are hampered because the minimal therapeutic conditions that would allow extracellular matrix remodeling and recovery of cells from stress are not known. Here, we employed a tetracycline-inducible system for expressing the R789C or R992C collagen II mutants, allowing us to decrease the production of mutant proteins by 25, 50, 75, or 100% with respect to their initial production. Through analysis of intracellular and extracellular parameters we have shown that affected cell/matrix systems are able to recover from mutation-induced aberrations only when 100% expression of mutant collagens is shut off, but not if the expression of small amounts of mutant molecules persists in the system. Our data suggest that efficient remodeling of tissues affected by the presence of thermolabile collagen mutants may depend on their complete elimination rather than on partial reduction.
PMCID: PMC4246508  PMID: 21472893
collagen mutations; collagen II; cartilage; spondyloepiphyseal dysplasia; endoplasmic reticulum stress
21.  Morquio A Syndrome-Associated Mutations: A Review of Alterations in the GALNS Gene and a New Locus-Specific Database 
Human Mutation  2014;35(11):1271-1279.
Morquio A syndrome (mucopolysaccharidosis IVA) is an autosomal recessive disorder that results from deficient activity of the enzyme N-acetylgalactosamine-6-sulfatase (GALNS) due to alterations in the GALNS gene, which causes major skeletal and connective tissue abnormalities and effects on multiple organ systems. The GALNS alterations associated with Morquio A are numerous and heterogeneous, and new alterations are continuously identified. To aid detection and interpretation of GALNS alterations, from previously published research, we provide a comprehensive and up-to-date listing of 277 unique GALNS alterations associated with Morquio A identified from 1,091 published GALNS alleles. In agreement with previous findings, most reported GALNS alterations are missense changes and even the most frequent alterations are relatively uncommon. We found that 48% of patients are assessed as homozygous for a GALNS alteration, 39% are assessed as heterozygous for two identified GALNS alterations, and in 13% of patients only one GALNS alteration is detected. We report here the creation of a locus-specific database for the GALNS gene ( that catalogs all reported alterations in GALNS to date. We highlight the challenges both in alteration detection and genotype–phenotype interpretation caused in part by the heterogeneity of GALNS alterations and provide recommendations for molecular testing of GALNS.
PMCID: PMC4238747  PMID: 25137622
MPS IVA; Morquio A; mucopolysaccharidosis type IVA; GALNS; lysosomal storage disorder
22.  Gene Conversion Violates the Stepwise Mutation Model for Microsatellites in Y-Chromosomal Palindromic Repeats 
Human Mutation  2014;35(5):609-617.
The male-specific region of the human Y chromosome (MSY) contains eight large inverted repeats (palindromes), in which high-sequence similarity between repeat arms is maintained by gene conversion. These palindromes also harbor microsatellites, considered to evolve via a stepwise mutation model (SMM). Here, we ask whether gene conversion between palindrome microsatellites contributes to their mutational dynamics. First, we study the duplicated tetranucleotide microsatellite DYS385a,b lying in palindrome P4. We show, by comparing observed data with simulated data under a SMM within haplogroups, that observed heteroallelic combinations in which the modal repeat number difference between copies was large, can give rise to homoallelic combinations with zero-repeats difference, equivalent to many single-step mutations. These are unlikely to be generated under a strict SMM, suggesting the action of gene conversion. Second, we show that the intercopy repeat number difference for a large set of duplicated microsatellites in all palindromes in the MSY reference sequence is significantly reduced compared with that for nonpalindrome-duplicated microsatellites, suggesting that the former are characterized by unusual evolutionary dynamics. These observations indicate that gene conversion violates the SMM for microsatellites in palindromes, homogenizing copies within individual Y chromosomes, but increasing overall haplotype diversity among chromosomes within related groups.
PMCID: PMC4233959  PMID: 24610746
Y chromosome; gene conversion; palindrome; microsatellite; stepwise mutation model; DYS385
23.  Molecular Analysis, Pathogenic Mechanisms, and Readthrough Therapy on a Large Cohort of Kabuki Syndrome Patients 
Human Mutation  2014;35(7):841-850.
Kabuki syndrome (KS) is a multiple congenital anomalies syndrome characterized by characteristic facial features and varying degrees of mental retardation, caused by mutations in KMT2D/MLL2 and KDM6A/UTX genes. In this study, we performed a mutational screening on 303 Kabuki patients by direct sequencing, MLPA, and quantitative PCR identifying 133 KMT2D, 62 never described before, and four KDM6A mutations, three of them are novel. We found that a number of KMT2D truncating mutations result in mRNA degradation through the nonsense-mediated mRNA decay, contributing to protein haploinsufficiency. Furthermore, we demonstrated that the reduction of KMT2D protein level in patients’ lymphoblastoid and skin fibroblast cell lines carrying KMT2D-truncating mutations affects the expression levels of known KMT2D target genes. Finally, we hypothesized that the KS patients may benefit from a readthrough therapy to restore physiological levels of KMT2D and KDM6A proteins. To assess this, we performed a proof-of-principle study on 14 KMT2D and two KDM6A nonsense mutations using specific compounds that mediate translational readthrough and thereby stimulate the re-expression of full-length functional proteins. Our experimental data showed that both KMT2D and KDM6A nonsense mutations displayed high levels of readthrough in response to gentamicin treatment, paving the way to further studies aimed at eventually treating some Kabuki patients with readthrough inducers.
PMCID: PMC4234006  PMID: 24633898
KMT2D; KDM6A; Kabuki syndrome; haploinsufficiency; readthrough
24.  Cadherin 5 is Regulated by Corticosteroids and Associated with Central Serous Chorioretinopathy 
Human Mutation  2014;35(7):859-867.
Central serous chorioretinopathy (CSC) is characterized by leakage of fluid from the choroid into the subretinal space and, consequently, loss of central vision. The disease is triggered by endogenous and exogenous corticosteroid imbalance and psychosocial stress and is much more prevalent in men. We studied the association of genetic variation in 44 genes from stress response and corticosteroid metabolism pathways with the CSC phenotype in two independent cohorts of 400 CSC cases and 1,400 matched controls. The expression of cadherin 5 (CDH5), the major cell–cell adhesion molecule in vascular endothelium, was downregulated by corticosteroids which may increase permeability of choroidal vasculature, leading to fluid leakage under the retina. We found a significant association of four common CDH5 SNPs with CSC in male patients in both cohorts. Two common intronic variants, rs7499886:A>G and rs1073584:C>T, exhibit strongly significant associations with CSC; P = 0.00012; odds ratio (OR) = 1.5; 95%CI [1.2;1.8], and P = 0.0014; OR = 0.70; 95%CI [0.57;0.87], respectively. A common haplotype was present in 25.4% male CSC cases and in 35.8% controls (P = 0.0002; OR = 0.61, 95% CI [0.47–0.79]). We propose that genetically predetermined variation in CDH5, when combined with triggering events such as corticosteroid treatment or severe hormonal imbalance, underlie a substantial proportion of CSC in the male population.
PMCID: PMC4215937  PMID: 24665005
CDH5; Cadherin 5; central serous chorioretinopathy; genetic association; retinal disease
25.  Combined NGS Approaches Identify Mutations in the Intraflagellar Transport Gene IFT140 in Skeletal Ciliopathies with Early Progressive Kidney Disease 
Human mutation  2013;34(5):714-724.
Ciliopathies are genetically heterogeneous disorders characterized by variable expressivity and overlaps between different disease entities. This is exemplified by the short rib-polydactyly syndromes, Jeune, Sensenbrenner, and Mainzer-Saldino chondrodysplasia syndromes. These three syndromes are frequently caused by mutations in intraflagellar transport (IFT) genes affecting the primary cilia, which play a crucial role in skeletal and chondral development. Here, we identified mutations in IFT140, an IFT complex A gene, in five Jeune asphyxiating thoracic dystrophy (JATD) and two Mainzer-Saldino syndrome (MSS) families, by screening a cohort of 66 JATD/MSS patients using whole exome sequencing and targeted resequencing of a customized ciliopathy gene panel. We also found an enrichment of rare IFT140 alleles in JATD compared with nonciliopathy diseases, implying putative modifier effects for certain alleles. IFT140 patients presented with mild chest narrowing, but all had end-stage renal failure under 13 years of age and retinal dystrophy when examined for ocular dysfunction. This is consistent with the severe cystic phenotype of Ift140 conditional knockout mice, and the higher level of Ift140 expression in kidney and retina compared with the skeleton at E15.5 in the mouse. IFT140 is therefore a major cause of cono-renal syndromes (JATD and MSS). The present study strengthens the rationale for IFT140 screening in skeletal ciliopathy spectrum patients that have kidney disease and/or retinal dystrophy.
PMCID: PMC4226634  PMID: 23418020
cilia; Jeune asphyxiating thoracic dystrophy; Mainzer-Saldino syndrome; IFT140; NGS

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