Background: Knowledge on clinical profiles of late-onset phenotypes of Fabry disease (FD) is essential to better define their natural history. Our study aims to demonstrate a founder effect of FD due to the GM gene mutation c.337T > C (p.F113L) in the Portuguese region of Guimaraes; and to characterize the clinical profile of this late-onset phenotype in a large cohort of genetically related adult patients, living in the same region. Methods and Results: FD screening was performed in 150 adult patients with hypertrophic cardiomyopathy (HCM) and found 25 Fabry patients (16.6%). The p.F113L mutation was found in 21 of them, leading to a genealogy study and haplotype analysis of the p.F113L patients. Genealogy research revealed a 12-generation family tree with a common ancestor to p.F113L patients, suggesting a founder effect that was supported by haplotype findings. Pedigree analysis was performed and 120 consecutive p.F113L patients underwent a pre-defined diagnostic evaluation of FD multiorgan involvement. This late-onset phenotype was characterized by common and/or potentially severe cardiac manifestations (left ventricular hypertrophy 40.8%, atrial fibrillation 5%, non-sustained ventricular tachycardia 12.5%, atrioventricular block 18.3%, bifascicular block 13.4%). Extracardiac manifestations included albuminuria > 30 mg/24 h 36.1%, chronic kidney disease >= G3 7.6%, brain white matter lesions 54.4%, stroke 3.3%, sensorineural deafness 44.5%, cornea verticillata 13.9%. Plasma lyso-GB3 was undetectable in females, regardless of clinical manifestations. Conclusion: A founder effect of FD due to p.F113L mutation was documented by genealogy and genetics in a Portuguese region. In this late-onset phenotype, although cardiac manifestations carry the highest prognostic impact, extracardiac involvement is common.

Background: As patients with different types of mucopolysaccharidosis (MPS) and mucolipidosis (ML) may present with overlapping clinical features - including coarse face, hepatosplenomegaly, bone dysplasia and claw-hand deformities, collectively also called 'MPS-like phenotype', enzymatic and/or molecular genetic analyses are indispensable for accurate diagnosis and applying specific therapy. In this prospective study, we screened patients with symptoms compatible with MPS for MPS I, II (males) and VI.Methods: Dried blood spots/specimens (DBS) were collected from 200 patients with an MPS-like phenotype and analysed for activities of alpha-iduronidase (IDUA), iduronate-2-sulphatase (IDS), and arylsulphatase B (ARSB), the enzymes deficient in mucopolysaccharidosis (MPS) type I, II and VI, respectively. For the samples with pathologic enzyme activity, mutational analysis was carried out using the same DBS.Results: Based on enzymatic analysis of 200 DBS samples, a total of 45 (22.5%) showed low activity; 17 for MPS I (8.5%), 11 for MPS II (5.5%) and 9 for MPS VI (4.5%). Enzyme activities were suggestive for ML II/III in 8 (4.0%) cases. For 41 (91.1%) samples, DNA could be extracted from the filter paper. Mutations were identified in 11 (64.7%), 11 (100%), 9 (100%) and 5 (62.5%) patients putatively diagnosed biochemically with MPS I, II, VI, and ML II/III, respectively.Conclusions: DBS enzymatic analysis can be used to diagnose MPS/ML. Initial results should be confirmed by a second enzyme assay and/or by molecular genetic testing. Given the advantages of DBS over other sample types in terms of ease of collection, storage and transportation, DBS are particularly useful for screening patients with an MPS-like phenotype in regions lacking specialised laboratories. In order to ascertain the diagnosis in a large number of cases, patients should be assessed in parallel for at least MPS I, II and VI.

Ocular signs in Fabry disease have generally been regarded to be primarily of diagnostic value. We explored whether ocular findings, alone or in particular in combination with the α-galactosidase A gene mutation, have predictive value for disease severity. Data from the Fabry Outcome Survey (FOS), a large, global database sponsored by Shire, were selected for adult patients who had undergone ophthalmological examination. Three ocular signs were assessed: cornea verticillata, tortuous conjunctival and/or retinal vessels, and cataract. Fabry disease severity was measured using FOS Mainz Severity Score Index and modifications thereof. Ophthalmological data were available for 1203 (699 female, 504 male) adult patients with eye findings characteristic of Fabry disease in 55.1%. Cornea verticillata had a similar distribution in women (51.1%) and men (50.8%), whereas tortuous vessels and Fabry cataract were somewhat more frequent in men than in women. Patients with cornea verticillata, selected as the principal ocular sign for this study, had more severe disease (median score, 20.0) versus those without ocular signs (11.0; P<0.001). This finding could be confirmed by applying age adjusted severity scores. Moreover, the prevalence of cornea verticillata was significantly higher in patients with null (male, 76.9%; female, 64.5%) and missense (male, 79.2%; female, 67.4%) mutations versus mild missense (male, 17.1%; female, 23.1%) and the p.N215S (male, 15.0%; female, 15.6%) mutations (P<0.01). Our analyses show a correlation between the prevalence of ocular changes in Fabry disease and disease severity. Consequently, information on ocular findings and α-galactosidase A gene mutation may help assess the risk for more severe Fabry disease. These observed findings are of notable clinical importance, as Fabry disease is characterized by high clinical course variability and only weak genotype-phenotype correlation at the individual patient level. Further confirmatory studies are needed.

Many neurodegenerative disorders present with sensory loss. In the group of hereditary sensory and autonomic neuropathies loss of nociception is one of the disease hallmarks. To determine underlying factors of sensory neurodegeneration we performed whole-exome sequencing in affected individuals with the disorder. In a family with sensory neuropathy with loss of pain perception and destruction of the pedal skeleton we report a missense mutation in a highly conserved amino acid residue of atlastin GTPase 3 (ATL3), an endoplasmic reticulum-shaping GTPase. The same mutation (p.Tyr192Cys) was identified in a second family with similar clinical outcome by screening a large cohort of 115 patients with hereditary sensory and autonomic neuropathies. Both families show an autosomal dominant pattern of inheritance and the mutation segregates with complete penetrance. ATL3 is a paralogue of ATL1, a membrane curvature-generating molecule that is involved in spastic paraplegia and hereditary sensory neuropathy. ATL3 proteins are enriched in three-way junctions, branch points of the endoplasmic reticulum that connect membranous tubules to a continuous network. Mutant ATL3 p.Tyr192Cys fails to localize to branch points, but instead disrupts the structure of the tubular endoplasmic reticulum, suggesting that the mutation exerts a dominant-negative effect. Identification of ATL3 as novel disease-associated gene exemplifies that long-term sensory neuronal maintenance critically depends on the structural organisation of the endoplasmic reticulum. It emphasizes that alterations in membrane shaping-proteins are one of the major emerging pathways in axonal degeneration and suggests that this group of molecules should be considered in neuroprotective strategies.

Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are major causes of blindness. They result from mutations in many genes which has long hampered comprehensive genetic analysis. Recently, targeted next-generation sequencing (NGS) has proven useful to overcome this limitation. To uncover "hidden mutations" such as copy number variations (CNVs) and mutations in non-coding regions, we extended the use of NGS data by quantitative readout for the exons of 55 RP and LCA genes in 126 patients, and by including non-coding 5' exons. We detected several causative CNVs which were key to the diagnosis in hitherto unsolved constellations, e.g. hemizygous point mutations in consanguineous families, and CNVs complemented apparently monoallelic recessive alleles. Mutations of non-coding exon 1 of EYS revealed its contribution to disease. In view of the high carrier frequency for retinal disease gene mutations in the general population, we considered the overall variant load in each patient to assess if a mutation was causative or reflected accidental carriership in patients with mutations in several genes or with single recessive alleles. For example, truncating mutations in RP1, a gene implicated in both recessive and dominant RP, were causative in biallelic constellations, unrelated to disease when heterozygous on a biallelic mutation background of another gene, or even non-pathogenic if close to the C-terminus. Patients with mutations in several loci were common, but without evidence for di- or oligogenic inheritance. Although the number of targeted genes was low compared to previous studies, the mutation detection rate was highest (70%) which likely results from completeness and depth of coverage, and quantitative data analysis. CNV analysis should routinely be applied in targeted NGS, and mutations in non-coding exons give reason to systematically include 5'-UTRs in disease gene or exome panels. Consideration of all variants is indispensable because even truncating mutations may be misleading.

Hereditary spastic paraplegias (HSP) are a heterogeneous group of neurological disorders. Insidiously progressive spastic weakness of the lower extremities is the common criterion in all forms described. Clinically, HSP is differentiated into pure (uncomplicated) and complex (complicated) forms. While pure HSP is predominantly characterized by signs and symptoms of pyramidal tract dysfunction, additional neurological and non-neurological symptoms occur in complicated forms. Autosomal dominant, autosomal recessive, and X-linked modes of inheritance have been described and at least 48 subtypes, termed SPG1-48, have been genetically defined. Although in autosomal dominant HSP families 50-60% of etiologies can be established by genetic testing, genotype predictions based on the phenotype are limited. In order to realize high-throughput genotyping for dominant HSP, we designed a resequencing microarray for six autosomal dominant genes on the Affymetrix CustomSEQ array platform. For validation purposes, 10 previously Sanger sequenced patients with autosomal dominant HSP and 40 positive controls with known mutations in ATL1, SPAST, NIPA1, KIF5A, and BSCL2 (32 base exchanges, eight small indels) were resequenced on this array. DNA samples of 45 additional patients with AD spastic paraplegia were included in the study. With two different sequencing analysis software modules (GSEQ, SeqC), all missense/nonsense mutations in the positive controls were identified while indels had a detection rate of only 50%. In total, 244 common synonymous single-nucleotide polymorphisms (SNPs) annotated in dbSNP (build 132) corresponding to 22 distinct sequence variations were found in the 53 analyzed patients. Among the 22 different sequence variations (SPAST n = 15, ATL1 n = 3, KIF5A n = 2, HSPD1 n = 1, BSCL2 n = 1, NIPA1 n = 0), 12 were rare variants that have not been previously described and whose clinical significance is unknown. In SPAST-negative cases, a genetic diagnosis could be established in 11% by resequencing. Resequencing microarray technology can therefore efficiently be used to study genotypes and mutations in large patient cohorts.

About 60-70 % of the heterozygotes of X-linked hypohidrotic ectodermal dysplasia (XLHED) show some clinical manifestations of the disease. Dental abnormalities are key diagnostic features and can be best evaluated at a young age. Compared to controls, carriers have a significantly higher frequency of agenesis of permanent teeth with persistence of deciduous teeth, small teeth resulting in gaps between the teeth, and peg-shaped teeth. Whole saliva flow seems to be reduced in carriers, whereas concentrations of most inorganic salivary constituents and total protein are higher than in controls. Craniofacial morphology of carriers shows some subtle deviations such as short and retrognatic maxilla, protruding lips, and a shorter total facial height compared to controls, findings that could be explained in part by hypodontia. Mild hypotrichosis and mild hypohidrosis are two further commonly seen carrier signs. No correlation has been found so far between the type of mutations, the patients’ phenotypes and disease severity. The mosaic-like distribution of normal and abnormal skin along the Blaschko lines is a typical consequence of the clonal inactivation of the X-chromosomes. While carriers with a highly skewed X-chromosome inactivation pattern may show pronounced clinical features due to the unequal expression of the two alleles (‘functional hemizygosity’), no correlation has been found between the severity of clinical symptoms and X-chromosome inactivation in leukocytes of carriers. Sporadic cases of males with XLHED and autosomal recessive HED (arHED) cannot be distinguished clinically. In contrast to heterozygotes of XLHED, who frequently show mild involvement, heterozygous parents of patients with arHED show no features of the disorder. However, because of the highly variable expression of XLHED in carriers, phenotypic assessment may result both in underdetection and false-positive diagnosis.

Posterior microphthalmos (MCOP) is a rare isolated developmental anomaly of the eye characterized by extreme hyperopia due to short axial length. The population of the Faroe Islands shows a high prevalence of an autosomal-recessive form (arMCOP) of the disease. Based on published linkage data, we refined the position of the disease locus (MCOP6) in an interval of 250 kb in chromosome 2q37.1 in two large Faroese families. We detected three different mutations in PRSS56. Patients of the Faroese families were either homozygous for c.926G>C (p.Trp309Ser) or compound heterozygous for c.926G>C and c.526C>G (p.Arg176Gly), whereas a homozygous 1 bp duplication (c.1066dupC) was identified in five patients with arMCOP from a consanguineous Tunisian family. In one patient with MCOP from the Faroe Islands and in another one from Turkey, no PRSS56 mutation was detected, suggesting nonallelic heterogeneity of the trait. Using RT-PCR, PRSS56 transcripts were detected in samples derived from the human adult retina, cornea, sclera, and optic nerve. The expression of the mouse ortholog could be first detected in the eye at E17 and was maintained into adulthood. The predicted PRSS56 protein is a 603 amino acid long secreted trypsin-like serine peptidase. The c.1066dupC is likely to result in a functional null allele, whereas the two point mutations predict the replacement of evolutionary conserved and functionally important residues. Molecular modeling of the p.Trp309Ser mutant suggests that both the affinity and reactivity of the enzyme toward in vivo protein substrates are likely to be substantially reduced.

Guidelines on diagnostics and therapy of Fabry disease have already been compiled in a number of European countries and are being prepared in others. A synthesis of these national guidelines seems to be a sensible option for preparing a prospective European consensus document in the not too distant future. While clinical diagnostics is extensively discussed in the various guidelines, at present there is no consensus on laboratory diagnostic tests for Fabry disease either at national level or at the level of the European Union. There is a widespread variation concerning the diagnostic value of the various methods, such as enzyme activity testing, gene analysis, biopsies, Gb3 measurement etc. and on what should be used and how they compare, if indeed they do. This results in lack of agreement regarding the clinical pathology of the condition. While experts throughout Europe agree on many aspects of the laboratory diagnosis of Fabry disease (for a recent review see Winchester and Young 2010), there are some specific issues which need further discussion before a general recommendation can be made. In order to promote this process, three one-and-a-half day ad hoc meetings* of European experts** were organized in 2009 with the aim of defining a consensus on laboratory diagnostics of Fabry disease that should allow for laboratories performing this diagnostic service to work to the same standards. Invitations to attend the meetings were based on, in addition to geographic considerations, outstanding scientific accomplishments in the field and/or active involvement in the laboratory diagnostics of Fabry disease. *‘Diagnostic use and value of Gb3 and lysoGb3 in Fabry disease’ (April 22-23, 2009, Bad Nauheim, Germany); ‘Diagnostic use and value of measuring alpha-galactosidase A activity in Fabry disease’ (July 16-17, 2009, Berlin, Germany); ‘Diagnostic use and value of mutations of the GLA gene in Fabry disease’ (November 3-4, 2009, Barcelona, Spain). **J.M.F.G. Aerts (Amsterdam, The Netherlands), M. Beck (Mainz, Germany), O. Bodamer (Salzburg, Austria), A. Cooper (Manchester, United Kingdom), A. Gal (Hamburg, Germany), D. Germain (Paris, France), R. Giugliani (Zürich, Switzerland), D. Hughes (London, United Kingdom), L. Kuchar (Prague, Czech Republic), J. Ledvinova (Prague, Czech Republic), Z. Lukacs (Hamburg, Germany), C. Navarro (Vigo, Spain), E. Paschke (Graz, Austria), M. Piraud (Lyon, France), A. Rolfs (Rostock, Germany), C. Sa Miranda (Porto, Portugal), M. van Slegtenhorst (Rotterdam, The Netherlands), H. Treslova (Prague, Czech Republic), M.T. Vanier (Lyon, France), F.W. Verheijen (Rotterdam, The Netherlands), B. Winchester (London, United Kingdom). Primary objectives of the initiative were (i) establishment of ‘gold standards’ and practical algorithms for diagnosis of Fabry disease, (ii) promotion of a concept of certification/accreditation for diagnostic and treatment centres of excellence, and (iii) provision of a platform for future treatment guidelines. In particular, three specific issues were selected by the organizers: the diagnostic utility of measurement of Gb3 and lysoGb3, evaluation of α-galactosidase A activity, and analysing mutations of the GLA gene in patients with Fabry disease. The most important goal of these workshops was to provide a platform for the experts to exchange ideas and experiences and share experimental data with each other, even if preliminary or yet unpublished, on the use, practical value, and significance of different laboratory approaches in the diagnostics of Fabry disease. All data were critically discussed by the panel and used to prepare this recommendation for the medical community. We hope that this document will serve as a starting point for further discussion that, in the end, should result in a consensus, provide essential guidance to physicians, and ensure a better differential and timely diagnosis of patients with Fabry disease. In normal human tissues, there are two lysosomal glycosidases with α-galactosidase activity towards synthetic substrates. α-Galactosidase A (α-GAL; EC 3.2.1.22) which is deficient in Fabry disease, acts on terminal α-galactosyl residues in glycosphingolipids, whereas the so called α-galactosidase B is an α-N-acetylgalactosaminidase (α-NAGAL; EC 3.2.1.49) that acts on natural substrates with terminal α-N-acetylgalactosaminyl residues and is defective in Schindler disease. α-Galactosidase A does not catalyse the hydrolysis of the natural substrates of α-NAGAL whereas α-NAGAL may act on some natural substrates with terminal α-galactosyl residues (Clark and Garman 2009). As both enzymes act on synthetic α-galactoside substrates, α-N-acetylgalactosamine, a specific inhibitor of α-NAGAL is added to assays of α-galactosidase A activity for the diagnosis of Fabry disease. The genes (GLA and NAGA) encoding α-GAL and α-NAGAL are on chromosomes Xq22.1 and 22q13, respectively. Although the two genes show considerable homology, as they evolved from a common ancestral precursor (Clark and Garman 2009), α-galactosidase A and α-NAGAL have distinct physicochemical properties, and antibodies raised against one do not cross-react with the other. α-Galactosidase A is a typical lysosomal hydrolase with optimal activity towards natural and synthetic substrates at pH 3.8–4.6. It is a glycoprotein and is transported to the lysosomes via the mannose-6-phosphate pathway. It is synthesized as a precursor of 50 kDa and is processed to a mature lysosomal form of 46 kDa by partial proteolysis and modification of its carbohydrates. The enzyme is a homodimer with an active site in each of the monomers. Hydrolysis of its natural substrates in vivo requires saposin B whereas that in vitro requires the addition of a detergent, usually sodium taurocholate. Patients with a genetic deficiency of saposin B also accumulate the substrates for α-galactosidase A, as in Fabry disease. The three-dimensional structure of recombinant human α-galactosidase A has been determined by X-ray crystallography at a resolution of 3.25 Å, and used to understand the effects of mutations on the structure and function of the enzyme (Garman and Garboczi 2004, Garman 2007). After thorough clinical evaluation, determination of α-galactosidase A activity is the first step in the laboratory diagnosis of a patient suspected of having Fabry disease, unless there is a known familial GLA mutation, for which DNA analysis is straightforward (see later). The activity can be determined in various materials, such as plasma, leukocytes, fibroblasts, or dried blood spots (DBS); the assay is rapid, reliable, and cost-effective. Currently the assay of α-galactosidase A activity in leukocytes represents the diagnostic ‘gold-standard’. A skin biopsy is rarely taken for initial diagnosis but studies on fibroblasts could be useful for molecular characterization of the enzyme deficiency. The most commonly used method of enzyme analysis is based upon cleavage of 4-methylumbelliferyl-α-D-galactoside, a synthetic fluorigenic substrate. Recently a substrate suitable to assay enzyme activity by electrospray ionization/tandem mass spectrometry (ESI/TMS) has also become available (Li et al. 2004, Zhang et al. 2010). The activity of α-galactosidase A should always be determined in the presence of α-N-acetylgalactosamine, which inhibits α-galactosidase B. In addition, another lysosomal enzyme, preferably β-galactosidase, should always be assayed to evaluate sample quality. As enzyme activity may diminish rapidly if the sample is inappropriately stored and/or transported, the diagnostic laboratory should be contacted in advance for information regarding appropriate sample storage and shipping conditions. About 2% of the population/Fabry patients carry the non-synonymous change c.937 G > T (p.Asp313Tyr). This variant has low α-galactosidase A activity in plasma compared to the wild-type and about 60% of the mean α-galactosidase A activity of the wild-type in cells. The p.Asp313Tyr variant is stable at lysosomal pH and is not disease-causing (pseudodeficiency). Males with the classic Fabry disease phenotype can be reliably diagnosed by detecting complete deficiency or only negligible (<5% of mean normal) α-galactosidase A activity. Single male patients with attenuated phenotypes may show considerable residual enzyme activities (e.g. Gaspar et al. 2010) that are still, however, well below the normal reference range. If initial enzyme activity analysis is performed on DBS of a male proband and shows pathologic values, a confirmatory assay, preferably on leukocytes, is recommended. For the index patient of any family with a biochemically proven α-galactosidase A deficiency, mutation analysis of the GLA gene should be offered. This test can confirm the diagnosis and may provide additional information for disease prognosis and therapy. It is essential when genetic counselling of family members is desired (see later). Identification of heterozygous females is not reliably made by the measurements of α-galactosidase A activity because of the significant levels of activity that may be present in these samples due to random X-inactivation. Thus for females, a number of complementary diagnostic approaches is used currently, including measurement of urinary/plasma storage products, and histology/electron microscopy. However, only the identification of a disease-relevant heterozygous GLA mutation allows definite diagnosis of carrier status. Neonatal screening for Fabry disease in males is technically feasible by measurement of α-galactosidase A activity in DBS using either the fluorigenic or mass spectrometric substrate and will detect cases both with complete deficiency and residual enzyme activity (Lin et al. 2009). High-throughput measurement of α-galactosidase A activity in leukocytes and DBS, for example by the technique of ESI/TMS, has been used to identify patients with Fabry disease in cohorts with advanced organ manifestations typical for Fabry disease (high-risk populations; for a recent review see Linthorst et al. 2010). It has been reported that β-glucuronidase activity is elevated in Fabry patients (Z. Lukacs, personal communication). Therefore the ratio of α-galactosidase A to β-glucuronidase activities may be helpful for increasing diagnostic discrimination, particularly for heterozygotes in these populations. Gb3 (Gb3Cer, globotriaosylceramide), also known as GL3 or CTH, is a substrate of α-galactosidase A and the main glycosphingolipid which accumulates when there is a deficiency of the enzyme. It may therefore be useful in diagnosis and assessment of disease burden. Deacylated Gb3, globotriaosylsphingosine (lysoGb3Cer or lysoGb3), is a minor metabolite recently identified in plasma of patients with Fabry disease. LysoGb3 might be more directly involved in the pathology and may also be useful in monitoring the disease (Aerts et al. 2008). Gb3 profiling (comparative analysis of various Gb3 isoforms) rather than measurement of the total urinary Gb3 concentrations, may represent a new diagnostic avenue in the future. For biochemical analysis, Gb3 is easily accessible in various body fluids, including plasma and urine, and there are a number of validated assays including HPLC, TLC and LC/MS. In general, Gb3 should be measured in urine rather than in plasma, whereas lysoGb3 should be determined in plasma (Young et al. 2005, Togawa et al. 2010). Samples need to be sent fresh to the laboratory or should be frozen and subsequently transported frozen. It is recommended that both the biochemical analysis of lysoGb3 and Gb3 in plasma and urine, respectively, as well as the metabolite analysis are performed in specialized centers with appropriate experience. Gb3 inclusions can also be detected in cells of various tissues and organs, e.g. by biopsy of the conjunctiva, skin, kidney or heart, by electron microscopy and histochemistry including immunohistochemistry. Plasma Gb3 levels do not correlate with disease manifestations: They are elevated in males with Fabry disease but are normal or only mildly elevated in females. Since Gb3 seems to be directly involved in the renal pathology of Fabry disease as a result of vascular compromise, podocyte toxicity, and focal tubular damage and sclerosis, quantification of urinary Gb3, that is derived primarily from tubular epithelial cells, is a possible diagnostic procedure for patients presenting with the classic Fabry disease phenotype (Sessa et al. 2003). A relationship between urinary Gb3 levels and Fabry disease severity as well as to therapeutic response has been demonstrated in a few studies (Whitfield et al. 2005, Banikazemi et al. 2007, Eng et al. 2001, Schiffmann et al. 2001). More recent studies suggest that there is no linear relationship between urinary Gb3 concentrations and end-organ effects of Fabry disease in male or in female patients. Furthermore it is currently unclear whether or not increased urinary Gb3 concentration can be taken as a specific marker for disease-related lysosomal storage. Whilst there is no doubt that Gb3 accumulates in patients with Fabry disease, from placental development onwards (Vedder et al. 2006), its role in the assessment or management of patients with this condition is contentious. While single assessments of Gb3 add only very little to the diagnosis of patients with biochemically and/or clinically ascertained Fabry disease, serial measurements of total urinary Gb3 (plasma lysoGb3) in the same individual may be used to monitor the progression of the pathological process. In male patients, a response to ERT is indicated by a decline of urinary Gb3, whereas an increase after prolonged enzyme administration may point to the existence of interfering antibodies against infused α-galactosidase A (Schiffmann et al. 2006, Vedder et al. 2007, Hughes et al. 2008). Analysis of total urinary Gb3 from a 12-24 h urine sample may be a diagnostic adjunct in females suspected of having Fabry disease when no male index patient is available for mutation analysis. In symptomatic males with Fabry disease, plasma lysoGb3 is elevated probably from an early age, and is strikingly high in adult male patients, although no correlation has been found between plasma lysoGb3 and disease manifestations (Aerts et al. 2008). In females, plasma lysoGb3 levels increase steadily and show a weak correlation with disease manifestation. Provided that comprehensive analyses of plasma lysoGb3 data from large cohorts of patients with Fabry disease confirm the preliminary observations, lysoGb3 may prove to be a promising novel diagnostic tool (Rombach et al. 2010). Where gene alterations are found with unclear functional or pathological implications in females, the assessment of Gb3 metabolites/isoforms (urinary Gb3 and plasma lysoGb3) is recommended. The GLA gene, encoding α-galactosidase A, contains seven exons ranging from 92 to 291 base pairs (bp) in length. The coding region consists of 1,290 bp and encodes a polypeptide of 429 amino acids, with the first 31 residues representing a signal sequence. As of 30th of June 2009, a total of 599 GLA sequence changes, including 435 probable pathogenic point mutations (missense, nonsense, and splice site) and 150 disease-causing ‘short length’ rearrangements (mainly deletions and duplications affecting less than 65 nucleotides) as well as 14 DNA polymorphisms have been reported (for a recent review see Gal 2010). Large rearrangements involving one or more exons or the whole GLA gene seem to be infrequent (ca. 5%) in patients with Fabry disease. Most of the GLA mutations found in patients with Fabry disease are novel (unique, ‘private’). The frequency of de novo mutations has been estimated to be 3-10%. There are a few reports of patients carrying two different, highly probable disease-causing GLA mutations on the same allele. In contrast, patients may carry one or more of the common GLA polymorphisms, including several non-synonymous changes, or rare non-pathogenic variants, in addition to their disease-causing mutation. To date, no experimentally validated data have been presented that any of these latter sequence variants, alone or in combination, are disease-causing. As a practical approach one can define two classes of mutations to assess their pathogenic significance. Class 1 mutations are predicted to have high probability of causing disease because they create a premature stop codon (nonsense or frame-shift mutations) with the consequent loss of the protein #Affect the evolutionarily conserved splice-site dinucleotides at the beginning or the end of one of the 6 GLA introns and interfere with the proper splicing of the GLA mRNA, or severely disrupt the coding sequence by a large rearrangement. Sequence changes resulting in a catalytically inactive (non-functional) enzyme, for example missense mutations affecting one of the 15 residues in the enzyme active site, or one of the 8 cysteines essential for proper three-dimensional protein folding, are also class 1 mutations. Finally, recurrent disease-causing mutations and the growing number of GLA mutations that have been shown to result in non-functional enzyme in in vitro expression studies in cells in cultures transduced with the relevant mutant cDNA also belong to this group. It has been suggested that the pathology of some ‘simple’ disease-causing mutations might occasionally be complex. Indeed, some DNA changes thought to be missense mutations (e.g. p.Ser65Thr, p.Gly183Ser, p.Lys213Asn, or p.Met267Ile) seem to interfere with normal splicing of the GLA mRNA (Lai et al. 2003). Similarly, two deep intronic point mutations (c.639 + 861 C > T and c.639 + 919 G > A) apparently result in α-galactosidase A deficiency by causing complex changes in the pattern of splicing (Ishii et al. 2002, Filoni et al. 2008). About 50% of males with enzymatically proven Fabry disease carry a Class 1 mutation. Mutations that have been found in males with normal α-galactosidase A activities or with somewhat decreased enzyme activities, which are still, however, well above the pathologic range of values, are called Class 2 mutations and are almost certainly non-pathogenic. These include polymorphisms or sequence changes, e.g. c.937 G > T (p.D313Y) described as pseudodeficiency alleles. Routine mutation analysis of the GLA gene consists of sequencing of the coding region and exon-intron boundaries. The interpretation of gene alterations should be performed by an expert and genetic counselling should be offered. In more than 97% of males with pathologic α-galactosidase A activities, a sequence variant (Class 1 mutations or yet unclassified sequence changes) can be detected by routine mutation analysis. The identification of Class 1 mutations is considered a very useful and independent confirmation of the biochemical (and clinical) diagnosis. A small number of mutations, in particular exon-spanning duplications or inversions and deep intronic mutations, may escape detection by using the above method and can only be identified by more sophisticated procedures, such as the analysis of the mRNA or MLPA (multiplex ligation-dependent probe amplification). However, even if no highly probable disease-causing mutation is found in a male patient with an α-galactosidase A deficiency, the diagnosis of Fabry disease remains valid. For mutations not categorized as Class 1 or Class 2, a thorough clinical, biochemical and genetic analysis of the patient and his family may allow designation of the mutation as disease-causing or not. Analysis of evolutionary conservation of the relevant sequence and its absence in a large cohort of unaffected individuals may provide additional arguments for a pathogenic relevance of a change. An easily accessible and quality-controlled gene-specific mutation database would be an invaluable tool both for physicians treating patients, genetic counselors, and scientists involved in DNA diagnostics or research. Both Class 1 and Class 2 mutations also occur in females. Since enzyme activity measurements do not reliably detect heterozygotes, and many of the clinical features of Fabry disease are frequently observed in the general population, DNA diagnostics is much less efficient in identifying heterozygotes among women with clinical suspicion for Fabry disease than in confirming diagnosis in males with enzymatically proven Fabry disease. Carriers in families with Fabry disease can be identified by pedigree analysis and/or by showing that they have inherited the family-specific mutation. If bidirectional sequencing of the coding region and exon-intron boundaries do not reveal heterozygosity for a Class 1 mutation in a female DNA sample, MLPA may help to pick up the few cases of large DNA rearrangements in females, whereas the analysis of mRNA may be complicated by nonsense-mediated decay. In addition, a thorough clinical re-evaluation of the patient and additional biochemical tests are recommended. A careful analysis of the pedigree by a genetic counselor should also be offered, keeping in mind that nine out of ten patients with Fabry disease should have a positive family history for the trait. In females with typical signs and symptoms of Fabry disease but without positive family history, only the identification of a disease-relevant heterozygous GLA mutation allows definite diagnosis of carrier status. Conclusions (Fig. 1): (i) Thorough analysis of the patient's family and medical history together with a clinical examination are essential prior to laboratory testing. (ii) Diagnostic tests should be done in laboratories with appropriate quality control schemes, experience, and sample load. (iii) Analysis of α-galactosidase A activity is the standard diagnostic test of Fabry disease in males. Molecular analysis of the GLA gene is necessary to diagnose heterozygotes. Algorithm for the laboratory diagnosis of (a) male and (b) female patients with Fabry disease. The flow charts represent summaries of the discussion and conclusions in the text, with extra clarification in points 1-6. 1) Demonstration of a deficiency of α-galactosidase A in leukocytes and plasma from the same blood sample is supporting evidence for a diagnosis of Fabry disease. 2) Variants of Fabry disease with residual α-galactosidase A activity. 3) A patient with some symptoms of Fabry disease not due to deficiency of α-galactosidase A. 4) Class 1 mutation and mutations previously found in affected male or female Fabry patients. 5) Low α-galactosidase A activity can be suggestive of carrier status but not definitive. 6) To decide whether the proband has Fabry disease, all or some of the following investigations should be carried out: (a) re-evaluation of clinical presentation, (b) analysis of family pedigree, (c) measurement of urinary Gb3, and (d) in absence of a Class 1 mutation, any sequence change detected in the GLA gene must be expressed in vitro to investigate its effect on activity and/or structure of enzyme The European Consensus on Diagnostics in Fabry Disease was sponsored by an unrestricted education grant generously awarded by The Center for Extramural Clinical Research and Education of Shire Human Genetic Therapies, Inc. This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Name of the disease (synonyms) Fabry disease, Anderson-Fabry disease. OMIM# of the disease 301500. Name of the analysed gene(s) or DNA/chromosome segments Alpha-galactosidase A, GLA. OMIM# of the gene(s) 300644. Review of the analytical and clinical validity as well as of the clinical utility of DNA-based testing for mutations in the GLA gene in diagnostic, predictive and prenatal settings for Fabry disease and for risk assessment in patients' relatives.

Purpose: Although Fabry disease is X linked and considered to affect primarily male hemizygotes, female heterozygotes may experience all the signs and symptoms of this metabolic disorder. This prospective, single-center, open-label, clinical trial was performed to evaluate the long-term response of female patients with Fabry disease to enzyme replacement therapy. Methods: Symptomatic women (average age = 47 years) enrolled in this 4-year study were treated with agalsidase alfa (Replagal®, Shire HGT, Inc.) at a dose of 0.2 mg/kg, every other week for 4 years ( N = 36). Clinical and biochemical assessments were conducted at 12-month intervals. Results: The Mainz Severity Score Index, a measure of total disease burden, was significantly reduced after 12 months ( P < 0.01) of treatment and continuously improved over 4 years. Brief Pain Inventory “pain at its worst” score was reduced from 4.6 ± 2.9 at baseline to 3.3 ± 2.9 after 12 months ( P = 0.001) and remained reduced through 4 years. Mean left-ventricular mass decreased from 89.4 ± 29.3 g/m 2.7 at baseline to 66.5 ± 29.3 g/m 2.7 after 12 months ( P < 0.001) and remained reduced through 4 years. Average kidney function (estimated glomerular filtration rate and proteinuria) remained constant during the study. No safety issues were identified. Conclusions: Long-term agalsidase alfa is effective and was well tolerated in women with Fabry disease.

Hereditary sensory and autonomic neuropathy type II (HSAN II) leads to severe mutilations because of impaired nociception and autonomic dysfunction. Here we show that loss-of-function mutations in FAM134B, encoding a newly identified cis-Golgi protein, cause HSAN II. Fam134b knockdown results in structural alterations of the cis-Golgi compartment and induces apoptosis in some primary dorsal root ganglion neurons. This implicates FAM134B as critical in long-term survival of nociceptive and autonomic ganglion neurons.

Mutations in TOPORS cause autosomal dominant retinitis pigmentosa (adRP). Examination of 160 adRP patients from continental Europe revealed nine exonic single nucleotide variants, eight of which reside in the coding region; three synonymous single nucleotide polymorphisms (SNPs; c.2319T > C, c.2991T > C and c.1560A > G), three nonsynonymous SNPs (c.58C > T/p.P20S, c.74C >G/p.S25W and c.1730C > A/p.S577Y) and two novel missense mutations (c.1205A > C/p.Q402P and c.1818T > G/p.S606R). Whether the latter two variants represent adRP causing mutations awaits further analysis.

Purpose To identify the gene mutations responsible for autosomal recessive retinitis pigmentosa (arRP) in Pakistani families. Methods A cohort of consanguineous families with typical RP phenotype in patients was screened by homozygosity mapping using microsatellite markers that mapped close to 21 known arRP genes and five arRP loci. Mutation analysis was performed by direct sequencing of the candidate gene. Results In two families, RP21 and RP53, homozygosity mapping suggested RHO, the gene encoding rhodopsin, as a candidate disease gene on chromosome 3q21. In six out of seven affected members from the two families, direct sequencing of RHO identified a homozygous c.448G>A mutation resulting in the p.Glu150Lys amino acid change. This variant was first reported in PMK197, an Indian arRP family. Single nucleotide polymorphism analysis in RP21, RP53, and PMK197 showed a common disease-associated haplotype in the three families. Conclusions In two consanguineous Pakistani families with typical arRP phenotype in the patients, we identified a disease-causing mutation (p.Glu150Lys) in the RHO gene. Single nucleotide polymorphism analysis suggests that the previously reported Indian family (PMK197) and the two Pakistani families studied here share the RHO p.Glu150Lys mutation due to a common ancestry.

Purpose Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28. Methods With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages. Results Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease. Conclusions The MDH1 gene is not the cause of RP28-linked arRP. Our experimental strategy shows that long-range genomic PCR followed by UHTs provides an excellent system to perform a thorough screening of candidate genes for hereditary retinal degeneration.

Purpose The goal of this study was to identify mutations in X-chromosomal genes associated with retinitis pigmentosa (RP) in patients from Germany, The Netherlands, Denmark, and Switzerland. Methods In addition to all coding exons of RP2, exons 1 through 15, 9a, ORF15, 15a and 15b of RPGR were screened for mutations. PCR products were amplified from genomic DNA extracted from blood samples and analyzed by direct sequencing. In one family with apparently dominant inheritance of RP, linkage analysis identified an interval on the X chromosome containing RPGR, and mutation screening revealed a pathogenic variant in this gene. Patients of this family were examined clinically and by X-inactivation studies. Results This study included 141 RP families with possible X-chromosomal inheritance. In total, we identified 46 families with pathogenic sequence alterations in RPGR and RP2, of which 17 mutations have not been described previously. Two of the novel mutations represent the most 3’-terminal pathogenic sequence variants in RPGR and RP2 reported to date. In exon ORF15 of RPGR, we found eight novel and 14 known mutations. All lead to a disruption of open reading frame. Of the families with suggested X-chromosomal inheritance, 35% showed mutations in ORF15. In addition, we found five novel mutations in other exons of RPGR and four in RP2. Deletions in ORF15 of RPGR were identified in three families in which female carriers showed variable manifestation of the phenotype. Furthermore, an ORF15 mutation was found in an RP patient who additionally carries a 6.4 kbp deletion downstream of the coding region of exon ORF15. We did not identify mutations in 39 sporadic male cases from Switzerland. Conclusions RPGR mutations were confirmed to be the most frequent cause of RP in families with an X-chromosomal inheritance pattern. We propose a screening strategy to provide molecular diagnostics in these families.