Characterization of new arylsulfatase A gene mutations reinforces genotype‐phenotype correlation in metachromatic leukodystrophy

Metachromatic Leukodystrophy (MLD) is a rare inherited lysosomal storage disorder caused by the deficiency of Arylsulfatase A (ARSA). The disease manifests itself with a broad spectrum of clinical variants, all characterized by progressive neurodegeneration in the central and peripheral nervous systems. The correlation between mutations in the ARSA gene, residual enzymatic activity associated with the mutated alleles and patients' phenotype, which has been extensively drawn for common ARSA mutations, has recently been expanded to rare ones. In this context, functional studies on the rare allelic variances acquire particular relevance for patients' prognostic evaluation. Here we have characterized eight newly identified ARSA mutations, through lentiviral vector‐based expression studies on cell lines and ARSA defective murine fibroblasts. In each case, the residual activity associated with the new mutant allele correlates well with the patient's phenotype. Therefore, our results confirm the importance of functional characterization of mutant alleles for a precise genotype‐based classification and definition of prognosis in MLD patients, which is particularly relevant for pre‐symptomatic diagnosis. © 2009 Wiley‐Liss, Inc.     

Molecular analysis and genotype‐phenotype correlation of Diamond‐Blackfan anemia

Diamond‐Blackfan anemia (DBA) features hypoplastic anemia and congenital malformations, largely caused by mutations in various ribosomal proteins. The aim of this study was to characterize the spectrum of genetic lesions causing DBA and identify genotypes that correlate with phenotypes of clinical significance. Seventy‐four patients with DBA from across Canada were included. Nucleotide‐level mutations or large deletions were identified in 10 ribosomal genes in 45 cases. The RPS19 mutation group was associated with higher requirement for chronic treatment for anemia than other DBA groups. Patients with RPS19 mutations, however, were more likely to maintain long‐term corticosteroid response without requirement for further chronic transfusions. Conversely, patients with RPL11 mutations were less likely to need chronic treatment. Birth defects, including cardiac, skeletal, hand, cleft lip or palate and genitourinary malformations, also varied among the various genetic groups. Patients with RPS19 mutations had the fewest number of defects, while patients with RPL5 had the greatest number of birth defects. This is the first study to show differences between DBA genetic groups with regards to treatment. Previously unreported differences in the rate and types of birth defects were also identified. These data allow better patient counseling, a more personalized monitoring plan, and may also suggest differential functions of DBA genes on ribosome and extra‐ribosomal functions.     

Comprehensive genotype‐phenotype correlation in AP‐4 deficiency syndrome; Adding data from a large cohort of Iranian patients

Mutations in adaptor protein complex‐4 (AP‐4) genes have first been identified in 2009, causing a phenotype termed as AP‐4 deficiency syndrome. Since then several patients with overlapping phenotypes, comprised of intellectual disability (ID) and spastic tetraplegia have been reported. To delineate the genotype‐phenotype correlation of the AP‐4 deficiency syndrome, we add the data from 30 affected individuals from 12 out of 640 Iranian families with ID in whom we detected disease‐causing variants in AP‐4 complex subunits, using next‐generation sequencing. Furthermore, by comparing genotype‐phenotype findings of those affected individuals with previously reported patients, we further refine the genotype‐phenotype correlation in this syndrome. The most frequent reported clinical findings in the 101 cases consist of ID and/or global developmental delay (97%), speech disorders (92.1%), inability to walk (90.1%), spasticity (77.2%), and microcephaly (75.2%). Spastic tetraplegia has been reported in 72.3% of the investigated patients. The major brain imaging findings are abnormal corpus callosum morphology (63.4%) followed by ventriculomegaly (44.5%). Our result might suggest the AP‐4 deficiency syndrome as a major differential diagnostic for unknown hereditary neurodegenerative disorders.     

SAAMP 2.0: An algorithm to predict genotype‐phenotype correlation of lysosomal storage diseases

Lysosomal storage diseases (LSDs) are a group of genetic disorders, resulting from deficiencies of lysosomal enzyme. Genotype‐phenotype correlation is essential for timely and proper treatment allocation. Recently, by integrating prediction outcomes of 7 bioinformatics tools, we developed a SAAMP algorithm to predict the impact of individual amino‐acid substitution. To optimize this approach, we evaluated the performance of these bioinformatics tools in a broad array of genes. PolyPhen and PROVEAN had the best performances, while SNP&GOs, PANTHER and I‐Mutant had the worst performances. Therefore, SAAMP 2.0 was developed by excluding 3 tools with worst performance, yielding a sensitivity of 94% and a specificity of 90%. To generalize the guideline to proteins without known structures, we built the three‐dimensional model of iduronate‐2‐sulfatase by homology modeling. Further, we investigated the phenotype severity of known disease‐causing mutations of the GLB1 gene, which lead to 2 LSDs (GM1 gangliosidosis and Morquio disease type B). Based on the previous literature and structural analysis, we associated these mutations with disease subtypes and proposed a theory to explain the complicated genotype‐phenotype correlation. Collectively, an updated guideline for phenotype prediction with SAAMP 2.0 was proposed, which will provide essential information for early diagnosis and proper treatment allocation, and they may be generalized to many monogenic diseases.     

Identification of 12 novel mutations and two new polymorphisms in the arylsulfatase A gene: Haplotype and genotype–phenotype correlation studies in spanish metachromatic leukodystrophy patients

Arylsulfatase A (ARSA) deficiency is the main cause of metachromatic leukodystrophy (MLD), a lysosomal disorder with no specific treatment. In view of the importance of genetic counseling, analyses of mutations and polymorphisms, including the ARSA pseudodeficiency allele, were carried out in 18 unrelated Spanish MLD patients. A systematic search allowed us to identify 100% of the alleles involving 17 different mutations, 12 of which are novel: G32S, L68P, R84W, P94A, G99V, P136S, W193X, H227Y, R288H, G308D, T327I, and IVS6‐12C→G. Two new polymorphisms, 2033C>T and 2059C>T, were identified in intron 6 which, in combination with two polymorphisms previously described (2161C>G and 2213C>G), gave rise to four different haplotypes in the control population. In addition, we also studied polymorphism 842G>T. Linkage disequilibrium was detected between mutations IVS2+1G→A, D255H, and T327I and specific haplotypes, suggesting a unique origin for these mutations. Moreover, mutation T327I was always associated with the T allele of the new rare variant A210A (893C>T). The distribution of mutation D255H (frequency 19.4%) among patients with different MLD clinical presentation revealed a clear genotype–phenotype correlation paralleling that reported for mutation IVS2+1G→A (frequency 25%). Among the novel mutations, only P136S and R288H occurred on a background of the ARSA pseudodeficiency allele. Screening 182 normal chromosomes identified a frequency of 8.8% of this allele; moreover, we identified two unrelated subjects with the polyA‐ mutation in the absence of the N350S mutation, and this infrequent haplotype reinforced the heterogeneity of conditions with ARSA deficiency. Hum Mutat 14:240–248, 1999. © 1999 Wiley‐Liss, Inc.     

Heterozygous SOX9 Mutations Allowing for Residual DNA‐binding and Transcriptional Activation Lead to the Acampomelic Variant of Campomelic Dysplasia

Campomelic dysplasia is a malformation syndrome with multiple symptoms including characteristic shortness and bowing of the long bones (campomelia). CD, often lethal due to airway malformations, is caused by heterozygous mutations in SOX9, an SRY‐related gene regulating testis and chondrocyte development including expression of many cartilage genes such as type II collagen. Male to female sex reversal occurs in the majority of affected individuals with an XY karyotype. A mild form without campomelia exists, in which sex‐reversal may be also absent. We report here two novel SOX9 missense mutations in a male (c.495C>G; p.His165Gln) and a female (c.337A>G; p.Met113Val) within the DNA‐binding domain leading to non‐lethal acampomelic CD. Functional analyses of mutant proteins demonstrate residual DNA‐binding and transactivation of SOX9‐regulated genes. Combining our data and reports from the literature we postulate a genotype‐phenotype correlation: SOX9 mutations allowing for residual function lead to a mild form of CD in which campomelia and sex reversal may be absent. © 2010 Wiley‐Liss, Inc.     

Pellagra‐like condition is xeroderma pigmentosum/Cockayne syndrome complex and niacin confers clinical benefit

An extremely rare pellagra‐like condition has been described, which was partially responsive to niacin and associated with a multisystem involvement. The condition was proposed to represent a novel autosomal recessive entity but the underlying mutation remained unknown for almost three decades. The objective of this study was to identify the causal mutation in the pellagra‐like condition and investigate the mechanism by which niacin confers clinical benefit. Autozygosity mapping and exome sequencing were used to identify the causal mutation, and comet assay on patient fibroblasts before and after niacin treatment to assess its effect on DNA damage. We identified a single disease locus that harbors a novel mutation in ERCC5, thus confirming that the condition is in fact xeroderma pigmentosum/Cockayne syndrome (XP/CS) complex. Importantly, we also show that the previously described dermatological response to niacin is consistent with a dramatic protective effect against ultraviolet‐induced DNA damage in patient fibroblasts conferred by niacin treatment. Our findings show the power of exome sequencing in reassigning previously described novel clinical entities, and suggest a mechanism for the dermatological response to niacin in patients with XP/CS complex. This raises interesting possibilities about the potential therapeutic use of niacin in XP.     

FOXE3 mutations: genotype‐phenotype correlations

Microphthalmia and anophthalmia (MA) are severe developmental eye anomalies, many of which are likely to have an underlying genetic cause. More than 30 genes have been described, each of which is responsible for a small percentage of these anomalies. Among these, is the FOXE3 gene, which was initially described in individuals with dominantly inherited anterior segment dysgenesis and, subsequently, associated with recessively inherited primary aphakia, sclerocornea and microphthalmia. In this work, we describe 8 individuals presenting with an MA phenotype. Among them, 7 are carrying biallelic recessive FOXE3 mutations and 2 of these have novel mutations: p.(Ala78Thr) and p.(Arg104Cys). The last of our patients is carrying in the heterozygous state the recessive p.(Arg90Leu) mutation in the FOXE3 gene. To further understand FOXE3 involvement in this wide spectrum of ocular anomalies with 2 different patterns of inheritance, we reviewed all individuals with ocular abnormalities described in the literature for which a FOXE3 mutation was identified. This review demonstrates that correlations exist between the mutation type, mode of inheritance and the phenotype severity. Furthermore, understanding the genetic basis of these conditions will contribute to overall understanding of eye development, improve the quality of care, genetic counseling and, in future, gene‐based therapies.     

Structural and mutational analysis of the xeroderma pigmentosum group D (XPD) gene

Individuals affected by the autosomal recessive disease xerodermapigmentosum (XP) are acutely sensitive to sunlight and predisposedto skin cancer on exposed areas. Cells cultured from XP patientsare both UV sensitive and defective in the nucleotide excisionrepair of damaged DNA. These cellular phenotypes are amenableto experimental strategies employing complementation, an approachpreviously used to demonstrate the correction of XP-D phenotypesfollowing the introduction of the XPD (ERCC2) gene. In the presentstudy, we have characterized the genomic organization of theXPD (ERCC2) gene and found it to be comprised of 23 exons. Thesedata were helpful in evaluating the functional integrity ofalleles in two XP-D cell lines. In cell line GM436 a C     

Lack of clear and univocal genotype‐phenotype correlation in familial Mediterranean fever patients: A systematic review

Familial Mediterranean fever (FMF) is the most common autosomal recessive autoinflammatory disease. To date, following the isolation of more than 280 MEFV sequence variants, the genotype‐phenotype correlation in FMF patients has been intensively investigated; however, an univocal and clear consensus has not been yet reached. Thus, the aim of this systematic review was to analyze the available literature findings in order to provide to scientific community an indirect estimation of the impact of genetic factors on the phenotypic variability of FMF. This systematic review has been conducted according to the Preferred Reporting Items for Systematic reviews and Meta‐Analysis (PRISMA) guidelines. The p.M694V mutation was reported to have a relatively severe clinical course, similarly, patients homozygous for M694I and M680I, or carrying a combination of both at codons 694 and 680, have a severe disease. Also, patients homozygous for M694V and V726A variants experienced more severe clinical picture. Conversely, heterozygous p.V726A and p.E148Q genotypes have been correlated with a milder disease course. At present, doubts remain on the potential pathogenic role of E148Q variant. The heterogenity in clinical FMF manifestations reflects the changes occuring in repertoire of mutations. We believe that clinical criteria and gene tests, enhancing each other, could better support the diagnosis of FMF.     

Spectrum of ATP7B mutations and genotype–phenotype correlation in large‐scale Chinese patients with Wilson Disease

Wilson disease (WD), an inherited disorder associated with ATP7B gene, has a wide spectrum of genotypes and phenotypes. In this study, we developed a rapid multiplex PCR‐MassArray method for detecting 110 mutant alleles of interest, and used it to examine genomic DNA from 1222 patients and 110 healthy controls. In patients not found to have any mutation in the 110 selected alleles, PCR‐Sanger sequencing was used to examine the ATP7B gene. We identified 88 mutations, including 9 novel mutations. Our analyses revealed p.Arg778Leu, p.Arg919Gly and p.Thr935Met showed some correlations to phenotype. The p.Arg778Leu was related to younger onset age and lower levels of ceruloplasmin (Cp) and serum copper, while p.Arg919Gly and p.Thr935Met both indicated higher Cp levels. Besides, the p.Arg919Gly was related to neurological subtype, and p.Thr935Met showed significant difference in the percentage of combined neurological and visceral subtype. Moreover, for ATP7B mutations, the more severe impact on ATP7B protein was, the younger onset age and lower Cp level presented. The feasibility of presymptomatic DNA diagnosis and predicting clinical manifestation or severity of WD would be facilitated with identified mutations and genotype–phenotype correlation precisely revealed in the study.     

The genotype–phenotype landscape of familial amyotrophic lateral sclerosis in Australia

Amyotrophic lateral sclerosis (ALS) is a clinically and genetically heterogeneous fatal neurodegenerative disease. Around 10% of ALS cases are hereditary. ALS gene discoveries have provided most of our understanding of disease pathogenesis. We aimed to describe the genetic landscape of ALS in Australia by assessing 1013 Australian ALS patients for known ALS mutations by direct sequencing, whole exome sequencing or repeat primed polymerase chain reaction. Age of disease onset and disease duration were used for genotype–phenotype correlations. We report 60.8% of Australian ALS families in this cohort harbour a known ALS mutation. Hexanucleotide repeat expansions in C9orf72 accounted for 40.6% of families and 2.9% of sporadic patients. We also report ALS families with mutations in SOD1 (13.7%), FUS (2.4%), TARDBP (1.9%), UBQLN2 (.9%), OPTN (.5%), TBK1 (.5%) and CCNF (.5%). We present genotype–phenotype correlations between these genes as well as between gene mutations. Notably, C9orf72 hexanucleotide repeat expansion positive patients experienced significantly later disease onset than ALS mutation patients. Among SOD1 families, p.I114T positive patients had significantly later onset and longer survival. Our report highlights a unique spectrum of ALS gene frequencies among patients from the Australian population, and further, provides correlations between specific ALS mutations with disease onset and/or duration.     

Dihydropteridine reductase deficiency: Physical structure of the QDPR gene,identification of two new mutations and genotype–phenotype correlations

Dihydropteridine reductase (DHPR) is an enzyme involved in recycling of tetrahydrobiopterin (BH₄), the cofactor of the aromatic amino acid hydroxylases. Its deficiency is characterized by hyperphenylalaninemia due to the secondary defect of phenylalanine hydroxylase and depletion of the neurotransmitters dopamine and serotonin, whose syntheses are controlled by tryptophan and tyrosine hydroxylases. The DHPR cDNA has been cloned and mapped on 4p15.3. In the present study we report the genomic structure of the DHPR gene (QDPR). This gene includes seven exons within a range of 84–564 bp; the corresponding introns are flanked by canonic splice junctions. We also present a panel of PCR primers complementary to intronic sequences that greatly facilitates amplification of the gene and provides a genomic DNA approach for mutation detection. We have used this approach to study six patients with DHPR deficiency. Four known mutations (G23D, H158Y, IVS5G + 1A, R221X) and two new mutations (Y150C and G218ins9bp) were found. The Y150C mutation was found in compound heterozygosity with G23D, a mutation always associated with a severe phenotype in homozygous patients. This patient has an intermediate phenotype (good response to monotherapy with BH₄). The mutant enzyme for Y150C was expressed in an E. coli system. Comparison of its kinetic parameters with those of the G23D mutant enzyme showed that it is not as effective as the wild-type enzyme, but is more active than the G23D mutant. This patient's intermediate phenotype is thus due to the mild DHPR mutation Y150C. Correlations between genotypes and phenotypes were also found for the other mutations. Hum Mutat 12:267–273, 1998. © 1998 Wiley-Liss, Inc.     

Chronological difference in walking impairment among Japanese group A xeroderma pigmentosum (XP-A) patients with various combinations of mutation sites

Almost all Japanese group A xeroderma pigmentosum (XP-A) patients have nonsense and/or nonsense codon-leading mutations in the XP group A (XPA) gene, and develop neurological abnormalities. Walking ability is one of the most important neuromuscular functions of the patients, because it determines their daily activities. We studied the correlation between the various combinations of mutations found by PCR-RFLP in Japanese XP-A patients and their chronological walking impairment. We classified these patients into six groups. Group I: A patient who was homozygous for the mutation at codon 116 in exon 3 (Type 1 mutation) could never walk unaided. Group III: Typical patients who were homozygous for the mutation at intron 3 (Type 2 mutation) could walk unaided till 7–16 years of age. Group V: Patients who were compound heterozygous for Type 2 mutation and for the mutation at codon 228 in exon 6 (Type 3 mutation) began to develop some walking difficulty at 5–13 years of age and became unable to walk at 25–28 years of age. Group VI: A patient who was homozygous for Type 3 mutation could walk unaided without any difficulty till the age of 21. The walking ability of group II and IV patients is not known yet.     

XPC branch‐point sequence mutations disrupt U2 snRNP binding,resulting in abnormal pre‐mRNA splicing in xeroderma pigmentosum patients

Mutations in two branch‐point sequences (BPS) in intron 3 of the XPC DNA repair gene affect pre‐mRNA splicing in association with xeroderma pigmentosum (XP) with many skin cancers (XP101TMA) or no skin cancer (XP72TMA), respectively. To investigate the mechanism of these abnormalities we now report that transfection of minigenes with these mutations revealed abnormal XPC pre‐mRNA splicing that mimicked pre‐mRNA splicing in the patients' cells. DNA oligonucleotide‐directed RNase H digestion demonstrated that mutations in these BPS disrupt U2 snRNP–BPS interaction. XP101TMA cells had no detectable XPC protein but XP72TMA had 29% of normal levels. A small amount of XPC protein was detected at sites of localized ultraviolet (UV)‐damaged DNA in XP72TMA cells which then recruited other nucleotide excision repair (NER) proteins. In contrast, XP101TMA cells had no detectable recruitment of XPC or other NER proteins. Post‐UV survival and photoproduct assays revealed greater reduction in DNA repair in XP101TMA cells than in XP72TMA. Thus mutations in XPC BPS resulted in disruption of U2 snRNP‐BPS interaction leading to abnormal pre‐mRNA splicing and reduced XPC protein. At the cellular level these changes were associated with features of reduced DNA repair including diminished NER protein recruitment, reduced post‐UV survival and impaired photoproduct removal. Hum Mutat 30:1–9, 2009. Published 2009 Wiley‐Liss, Inc.     

剪切修复基因XPD抑制Ox-LDL诱导人脐动脉平滑肌细胞的增殖

目的:探讨剪切修复基因——着色性干皮病D组基因(XPD)在氧化低密度脂蛋白(Ox-LDL)促血管平滑肌细胞增殖中的作用及机制。方法:将重组质粒pEGFP-N2/XPD利用脂质体转染人脐动脉平滑肌细胞(HUASMCs),实验分为空白对照组、空载质粒pEGFP-N2组、重组质粒pEGFP-N2/XPD组、Ox-LDL组、Ox-LDL+pEGFP-N2组和Ox-LDL+pEGFP-N2/XPD组。用MTT法和Ed U法测定各组细胞的增殖率;流式细胞术检测各组细胞周期分布;利用Western blot法检测XPD、caspase-3、Bcl-2和Bax的蛋白水平。结果:Western blot实验结果发现,与空白对照组相比,pEGFP-N2/XPD组的XPD表达增加(P0.05),表明转染成功;MTT和Ed U检测结果显示,pEGFP-N2/XPD组的细胞增殖率较空白对照组降低(P0.05);与Ox-LDL组比较,Ox-LDL+pEGFP-N2/XPD组细胞增殖明显被抑制(P0.05)。流式细胞术的检测结果显示,与空白对照组比较,pEGFP-N2/XPD组的S期细胞比例明显减少(P0.05),G0/G1期细胞比例明显增多(P0.05);与Ox-LDL组比较,Ox-LDL+pEGFP-N2/XPD组的S期细胞比例减少(P0.05),G0/G1期细胞比例明显增多(P0.05)。Western blot结果显示,与对照组比较,pEGFP-N2/XPD组的cleaved caspase-3和Bax蛋白水平增加(P0.05),Bcl-2蛋白表达降低(P0.05);与Ox-LDL组比较,Ox-LDL+pEGFP-N2/XPD组的cleaved caspase-3和Bax蛋白水平增加(P0.01),Bcl-2蛋白表达降低(P0.05)。结论:XPD能抑制HUASMCs的增殖并促其凋亡,还能抑制Ox-LDL的促HUASMCs增殖作用,有可能成为抗动脉粥样硬化治疗的靶点。     

Congenital central hypoventilation syndrome: genotype–phenotype correlation in parents of affected children carrying a PHOX2B expansion mutation

Parodi S, Vollono C, Baglietto MP, Balestri M, Di Duca M, Landri PA, Ceccherini I, Ottonello G, Cilio MR. Congenital central hypoventilation syndrome: genotype–phenotype correlation in parents of affected children carrying a PHOX2B expansion mutation. Congenital Central Hypoventilation Syndrome (CCHS) is a rare genetic disorder. Although most CCHS associated PHOX2B mutations occur de novo, about 10% of the cases are inherited from apparently asymptomatic parents, thus confirming variable expressivity and incomplete penetrance of PHOX2B mutations. Three asymptomatic parents of children affected with CCHS, and found to carry the same PHOX2B expansion mutations as their siblings, were studied by overnight polysomnography and somatic mosaicism analysis. In one case, significant sleep breathing control anomalies were detected, while the other two resulted in normal. In tissue‐specific allele studies, mosaicism with a comparatively low mutant allele proportion was showed in the two unaffected adult carriers. Accurate polysomnography and assessment of the degree of somatic mosaicism should be conducted in asymptomatic carriers of PHOX2B mutations, as they may unmask subclinical but significant anomalies