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.     

Identification of nine novel arylsulfatase a (ARSA) gene mutations in patients with metachromatic leukodystrophy (MLD)

Metachromatic leukodystrophy (MLD) is a rare autosomal recessive disorder caused by mutations of the arylsulfatase A (ARSA) gene. We have investigated more than fifty MLD patients using allele-specific PCR assays to detect the pseudodeficiency (PD) allele and several common MLD mutations, followed by comprehensive nucleotide sequencing of the ARSA gene to detect rare or private mutations. Here we report the identification of nine novel microlesions in the ARSA gene: five missense mutations (c.464C>T, c.542T>A, c.916T>C, c.973G>A, c.1286A>C), three frameshift mutations (c.205_206delTG, c.489_495del, c.1483_1486dup), and one splice donor site mutation (c.973+1G>A). Comprehensive mutation detection has facilitated carrier detection and prenatal diagnosis for several at-risk MLD families.     

Occurrence,distribution, and phenotype of arylsulfatase A mutations in patients with metachromatic leukodystrophy

Occurrence, distribution, and phenotype of arylsulfatase A (ASA) mutations were investigated in 27 patients with metachromatic leukodystrophy (MLD) from Central Europe, mainly from Austria (n = 15) and Poland (n = 9). Genomic DNA from leukocytes, fibroblasts, or paraffin-embedded, formalin-fixed brain or nerve tissue, respectively, was tested by natural or mutated primer-modulated PCR restriction, fragment length polymorphism for the eight most common European mutations: R84Q, S96F, 459+1G>A, I179S, A212V, 1204+1G>A, P426L, and 1401del11bp. The overall identification rate of unrelated MLD alleles was the highest, in adult (90%), medium in juvenile (50%), and lowest in late infantile (36%) MLD patients. The two common alleles, 459+1G>A and P426L, together accounted for 42% of all 50 unrelated MLD alleles investigated; I179S was observed in 6 of 50 MLD alleles (12%). Thus, I179S was far more frequent than hitherto thought and appears to be a third common mutation in Europe. Moreover, a different allelic distribution between Austrian and Polish juvenile patients was disclosed, indicating genetic heterogeneity of MLD even within Central Europe. The genotype-phenotype correlation suggested by Polten et al. [N Engl J Med 324:18–22, 1991] was not followed by all of our MLD patients. Moreover, some MLD patients with identical ASA mutations presented with different phenotypes. This may be due, at least in some cases, to the presence of an additional mutation on individual mutant alleles. Therefore, prediction of the clinical course from single mutation analysis is not possible. Am. J. Med. Genet. 69:335–340, 1997. © 1997 Wiley-Liss, Inc.     

Coincidence of two novel arylsulfatase a alleles and mutation 459+1G>A within a family with metachromatic leukodystrophy: Molecular basis of phenotypic heterogeneity

In a family with three siblings, one developed classical late infantile metachromatic leukodystrophy (MLD), fatal at age 5 years, with deficient arylsulfatase A (ARSA) activity and increased galactosylsulfatide (GS) excretion. The two other siblings, apparently healthy at 12½ and 15 years, respectively, and their father, apparently healthy as well, presented ARSA and GS values within the range of MLD patients. Mutation screening and sequence analysis disclosed the involvement of three different ARSA mutations being the molecular basis of intrafamilial phenotypic heterogeneity. The late infantile patient inherited from his mother the frequent 0‐type mutation 459+1G>A, and from his father a novel, single basepair microdeletion of guanine at nucleotide 7 in exon 1 (7delG). The two clinically unaffected siblings carried the maternal mutation 459+1G>A and, on their paternal allele, a novel cytosine to thymidine transition at nucleotide 2435 in exon 8, resulting in substitution of alanine 464 by valine (A464V). The fathers genotype thus was 7delG/A464V. Mutation A464V was not found in 18 unrelated MLD patients and 50 controls. A464V, although clearly modifying ARSA and GS levels, apparently bears little significance for clinical manifestation of MLD, mimicking the frequent ARSA pseudodeficiency allele. Our results demonstrate that in certain genetic conditions MLD‐like ARSA and GS values need not be paralleled by clinical disease, a finding with serious diagnostic and prognostic implications. Moreover, further ARSA alleles functionally similar to A464V might exist which, together with 0‐type mutations, may cause pathological ARSA and GS levels, but not clinical outbreak of the disease. Hum Mutat 13:61–68, 1999. © 1999 Wiley‐Liss, Inc.     

Two new polymorphisms in the arylsulfatase A gene and their haplotype associations with normal,metachromatic leukodystrophy and pseudodeficiency alleles

Linkage disequilibrium exists between metachromatic leukodystrophy (MLD) and pseudodeficiency mutations and selected polymorphisms within the arylsulfatase A gene. We have identified 2 new polymorphic NlaIII sites, NlaIII¹ and NlaIII², in the gene that, when used in combination with the known BsrI and BamHI polymorphisms, extends the haplotype associations of the pseudodeficiency and the most common infantile onset MLD alleles. Fixed haplotypes have also been established for 3 other recurring MLD mutations, ala212val, pro4261eu, and thr274met. The NlaIII² site is relatively rare and was found only in association with the pseudodeficiency variant carrying the glycosylation site mutation alone. Am. J. Med. Genet. 73:32–35, 1997. © 1997 Wiley-Liss, Inc.     

Molecular and clinical consequences of novel mutations in the arylsulfatase A gene

Metachromatic leukodystrophy (MLD), a severe neurodegenerative metabolic disorder, is caused by deficient activity of arylsulfatase A (ARSA; EC 3.1.6.8), which leads to a progressive demyelinating process in central and peripheral nervous systems. In this study, a DNA sequence analysis was performed on six Polish patients with different types of MLD. Six novel mutations were identified: one nonsense (p.R114X), three missense (p.G122C, p.G293C, p.C493F) and two frameshift mutations (g.445_446dupG and g.2590_2591dupC). Substitutions p.G293C and p.C493F and duplication g.445_446dupG caused a severe reduction of enzyme activity in transient transfection experiments on mammalian cells (less than 1% of wild-type (WT) ARSA activity). Duplication 2590_2591dupC preserved low-residual ARSA activity (10% of WT ARSA). In summary, the novel MLD-causing mutations in the exons 2, 5 and even in 8 of the ARSA gene described here can be classified as severe type 0, leading in homozygosity to the late infantile form MLD. Growth retardation, delayed motor development, gait disturbances, tonic–clonic seizures and non-epileptic muscle spasms were the first onset symptoms in patients with late infantile form of MLD. In individual with juvenile type MLD gait disturbances evidenced the onset of the disease, while in a patient with late juvenile MLD, difficulties at school were displayed.     

Metachromatic leukodystrophy: Identification of the first deletion in exon 1 and nine novel point mutations in the arylsulfatase A gene

Metachromatic leukodystrophy (MLD), a lysosomal storage disease caused by the deficiency of arylsulfatase A (ASA), is inherited as an autosomal recessive trait, and its frequency is estimated to be 1 in 40,000 live births. Genomic DNA from 21 MLD patients (14 late-infantile and 7 juvenile cases) was amplified in four overlapping PCR fragments and tested by allele-specific oligonucleotide (ASO) for the two common mutations 459+1G→A and P426L. These mutations were found in only 28.6% of the alleles studied. The remaining alleles were analyzed by chemical mismatch cleavage (CMC) and automatic sequencing. In addition to five previously reported mutations (459+1G→a, A212V, R244C, R390W, P426L), 10 novel mutations were identified: 9 missense mutations (S95N, G119R, D152Y, R244H, S250Y, A314T, R384C, R496H, K367N) and one 8 bp deletion in exon 1, the first mutation reported in this exon. These methods allowed us to identify 76% of the alleles tested. Genotype-phenotype correlations could be established for some of these mutations. These results confirm the heterogeneity of mutations causing MLD and suggest that CMC is a reliable and informative screening method for point mutation detection in the arylsulfatase A gene. Hum Mutat 9:234–242, 1997. © 1997 Wiley-Liss, Inc.     

Characterization of two arylsulfatase A missense mutations D335V and T274M causing late infantile metachromatic leukodystrophy

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulfatase A. We describe a novel missense mutation in exon 6 causing the substitution of Asp335 by Val. In transient transfections no enzyme activity could be expressed from the arylsulfatase A cDNA carrying this mutation. Examination of the effects of the mutation in cells stably overexpressing the mutant enzyme revealed, that the mutant enzyme is catalytically inactive and degraded in an early biosynthetic compartment. We have also investigated the effects of a previously identified mutation (T274M). The specific catalytic activity of the Met274 substituted arylsulfatase is reduced to about 35% of the normal enzyme when measured with an artificial substrate. Most of this enzyme is also degraded in an early biosynthetic compartment. © 1996 Wiley-Liss, Inc.     

Late juvenile metachromatic leukodystrophy (MLD) in three patients with a similar clinical course and identical mutation on one allele

Metachromatic leukodystrophy (MLD) is an autosomal, recessively inherited, lysosomal storage disease caused by arylsulfatase A (ASA) activity deficit. Arylsulfatase A initiates the degradation of sulfatide (cerebroside sulfate), which is an essential component of myelin. The main clinical symptoms are caused by progressive demyelination. At least 37 MLD-related ASA mutations are known to date. I179S (E3P799) is a disease-related mutation, described for the first time by Fluharty in 1991. This aberration appears to substantially reduce, but not completely eliminate ASA activity, and was detected in individuals with late-onset (juvenile or adult) forms of MLD. This paper deals with the peculiar clinical course in three unrelated juveniles with late-onset MLD carrying the I179S mutation on one allele. In the three described patients with the I179S mutation, psychiatric disturbances and intellectual impairment dominated the clinical picture, while the neurological lesions progressed more slowly. Although the symptoms appeared rather early, making it possible to classify this as the juvenile type of MLD, the clinical picture was more that of the adult type. Although the mutations on the second allele in our patients are unknown, one can speculate, that the mutation I179S plays an important role in the characteristic clinical course (psychiatric impairment, slower neurological deterioration, but relatively early onset). It seems that I179S mutation on one allele with another mutation on the other allele reduces ASA activity, but the enzyme can still cope with a part of the substrate influx, leading to late-juvenile-onset MLD with such strikingly similar phenotypes remaining a little bit of the adult (psychiatric) type. This could be one more argument in favour of phenotype-genotype correlation in patients with MLD.     

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.     

A T > C transition causing a Leu > Pro substitution in a conserved region of the arylsulfatase A gene in a late infantile metachromatic leukodystrophy patient

A T >C transition (L428P) was detected in the arylsulfatase A alleles of a late infantile metachromatic leukodystrophy patient. The mutation causes a Leu > Pro substitution in exon 8. It lies in a region conserved among arylsulfatases. The mutation was not detected in 37 other patients and in 57 normal controls.     

Identification and characterization of 15 novel GALC gene mutations causing Krabbe disease

The characterization of the underlying GALC gene lesions was performed in 30 unrelated patients affected by Krabbe disease, an autosomal recessive leukodystrophy caused by the deficiency of lysosomal enzyme galactocerebrosidase. The GALC mutational spectrum comprised 33 distinct mutant (including 15 previously unreported) alleles. With the exception of 4 novel missense mutations that replaced evolutionarily highly conserved residues (p.P318R, p.G323R, p.I384T, p.Y490N), most of the newly described lesions altered mRNA processing. These included 7 frameshift mutations (c.61delG, c.408delA, c.521delA, c.1171_1175delCATTCinsA, c.1405_1407delCTCinsT, c.302_308dupAAATAGG, c.1819_1826dupGTTACAGG), 3 nonsense mutations (p.R69X, p.K88X, p.R127X) one of which (p.K88X) mediated the skipping of exon 2, and a splicing mutation (c.1489+1G>A) which induced the partial skipping of exon 13. In addition, 6 previously unreported GALC polymorphisms were identified. The functional significance of the novel GALC missense mutations and polymorphisms was investigated using the MutPred analysis tool. This study, reporting one of the largest genotype-phenotype analyses of the GALC gene so far performed in a European Krabbe disease cohort, revealed that the Italian GALC mutational profile differs significantly from other populations of European origin. This is due in part to a GALC missense substitution (p.G553R) that occurs at high frequency on a common founder haplotype background in patients originating from the Naples region.     

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.     

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.     

Dental and extra‐oral clinical features in 41 patients with WNT10A gene mutations: A multicentric genotype–phenotype study

WNT10A gene encodes a canonical wingless pathway signaling molecule involved in cell fate specification as well as morphogenetic patterning of the developing ectoderm, nervous system, skeleton, and tooth. In patients, WNT10A mutations are responsible for ectodermal‐derived pathologies including isolated hypo‐oligodontia, tricho‐odonto‐onycho‐dermal dysplasia and Schöpf‐Schulz‐Passarge syndrome (SSPS). Here we describe the dental, ectodermal, and extra‐ectodermal phenotypic features of a cohort of 41 patients from 32 unrelated families. Correlations with WNT10A molecular status (heterozygous carrier, compound heterozygous, homozygous) and patient's phenotypes were performed. Mild to severe oligodontia was observed in all patients bearing biallelic WNT10A mutations. However, patients with compound heterozygous mutations presented no significant difference in phenotypes compared with homozygous individuals. Anomalies in tooth morphology were frequently observed with heterozygous patients displaying hypodontia. No signs of SSPS, especially eyelids cysts, were detected in our cohort. Interestingly, extra‐ectodermal signs consisted of skeletal, neurological and vascular anomalies, the latter suggesting a wider phenotypic spectrum associated with WNT10A mutations. Indeed, the Wnt pathway plays a crucial role in skeletal development, lipid metabolism, and neurogenesis, potentially explaining patient's clinical manifestations.     

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.     

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