Mutation identification of Fabry disease in families with other lysosomal storage disorders

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1 Clin Genet 2013: 84: Printed in Singapore. All rights reserved Short Report 2012 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd CLINICAL GENETICS doi: /cge Mutation identification of Fabry disease in families with other lysosomal storage disorders Zampetti A, Fania L, Antuzzi D, Giurdanella F, Gnarra M, Bertola F, Lualdi S, Filocamo M, Morrone A, Feliciani C. Mutation identification of Fabry disease in families with other lysosomal storage disorders. Clin Genet 2013: 84: John Wiley & Sons A/S. Published by John Wiley & Sons Ltd, 2012 Fabry disease (FD) is an X-linked lysosomal storage disorder (LSD) caused by the deficiency of the enzyme α-galactosidase. It exhibits a wide clinical spectrum that may lead to a delayed or even missed diagnosis and the real incidence can be underestimated. We report the cases of two unrelated Italian families in whom FD was incidentally diagnosed in two females. In both families, the risk for other lysosomal disorders was known from other members affected by fucosidosis or mucopolysaccharidosis I Hurler/Scheie. Some subjects were simultaneously heterozygous for Fabry and the other lysosomal deficiency. Our study shows that the risk for more than one LSDs can occur in a family pedigree. The diagnosis of Fabry in female probands represents a diagnostic challenge, as symptoms and signs can be variably present because of the random X-chromosome inactivation. Conflictofinterest The authors declare that they have no conflict of interest. A Zampetti a, L Fania a, D Antuzzi b, F Giurdanella a, M Gnarra a,fbertola c, S Lualdi d, M Filocamo d, A Morrone e,f and C Feliciani a a Department of Dermatology, b Department of Pediatric Sciences, Policlinico A. Gemelli, Università Cattolica del Sacro Cuore, Rome, Italy, c Consortium for Human Molecular Genetics, University of Milano-Bicocca, Monza, Italy, d Centro di Diagnostica Genetica e Biochimica delle Malattie Metaboliche, Istituto G. Gaslini, Genova, Italy, e Laboratory for Molecular and Cell Biology of Neurologic and Metabolic Disorders, Paediatric Neurology Unit, Meyer Children s Hospital, Florence, Italy, and f Department of Sciences for Woman and Child s Health, University of Florence, Florence, Italy Key words: Fabry fucosidosis GLA mutation mucopolysaccharidosis Corresponding author: Anna Zampetti, MD, PhD, Department of Dermatology, Policlinico A. Gemelli, Università Cattolica del Sacro Cuore, Largo Gemelli 8, Rome, Italy. Tel.: ; fax: ; anna.zampetti@gmail.com Received 4 July 2012, revised and accepted for publication 29 November 2012 Lysosomal storage disorders (LSDs) are a group of at least 50 different genetic diseases resulting from the defective function of specific lysosomal enzymes and, in a few cases, of non-enzymatic lysosomal or nonlysosomal proteins. To date, worldwide epidemiological data on LSDs are unavailable or limited to specific populations. The overall incidence of LSDs in Australia is estimated to be 1:5000 1:8000 (1). Fabry disease (FD; MIM# ) is the second most prevalent lipid storage disorder after Gaucher disease. It is currently considered highly underestimated with an average diagnostic delay of about 10 years from the onset of symptoms (2). Characterized by an X-linked recessive inheritance, FD is caused by the deficiency of α-galactosidase A (GAL-A) activity leading to a progressive accumulation of globotriaosylceramide (Gb-3) and related glycosphingolipids within the lysosomes of different cell types, especially the endothelial cells. The residual enzyme activity appears to influence the clinical manifestations, depending on the type of GLA gene 281

2 Zampetti et al. mutation, but a clear genotype phenotype correlation has not been showed (3). First symptoms usually start during childhood with gastrointestinal disturbances and development of acroparesthesia, fever and diminished sweating (4). The pathognomonic cornea verticillata is present in 62% of male and 72% of female Fabry children; hearing impairment is also quite frequent with tinnitus and hearing loss reported in 25% of male and 33% of female affected children (5, 6). Cutaneous signs include angiokeratoma on the bathing trunk area and on the mucosae, telangiectasias and limbs lymphedema (7). The so-called Fabry facies is predominantly observed in males, but occasionally in females (8). Cardiovascular, renal and central nervous system involvement are responsible for the reduction of life expectancy in Fabry patients: years in males and 70 years in females (2). Occasionally, heterozygous female Fabry patients can be severely affected similar to hemizygous males, because of X-random inactivation (9). Fucosidosis (MIM# ) is an autosomic recessive LSD resulting from the deficiency of the lysosomal enzyme α-fucosidase. Mutations of the FUCA1 gene (MIM# ) on chromosome 1p34.1-1p36.1, containing eight exons spanning 23 kb, induce defective activity of α-fucosidase and impaired oligosaccharide cleavage with generalized accumulation of α- l-fucosylated glycoproteins, glycolipids, and oligosaccharides in tissues. Angiokeratoma corporis diffusum, coarse facial features, delayed intellectual and motor development with progressive neurological decline and musculoskeletal alterations are usually observed in the type I form and are absent in the adult mild form of type II fucosidosis (10). Mucopolysaccharidosis type I (MPS I; MIM# ) is an autosomic recessive LSD due to mutations in IDUA gene (MIM# ) located on chromosome 4p16.3. Deficiency of α-l-iduronidase results in a defective lysosomal degradation of mucopolysaccharides with high urine levels of glycosaminoglycans. MPS I clinical phenotype can include gargoyle facies, cardiac, visceral skeletal and neurological involvement. Three distinct phenotypes are classically identified: (i) severe (Hurler syndrome) with early onset of symptoms, survival no more than 10 years and mental retardation; (ii) intermediate (Hurler/Scheie syndrome) when onset of symptoms is between 1 and 6 years, variable survival and absent or mild mental retardation; and (iii) attenuated (Scheie syndrome) when symptoms become apparent after the age of 5, survival is normal and mental retardation is never present (11). We now report the diagnosis of FD incidentally achieved in two female patients belonging to two distinct Italian families at risk for fucosidosis and MPS I. Materials and methods Family pedigrees were built by collecting information from the two female probands and their relatives. Following ethical guidelines, the blood samples were collected for molecular analyses after having obtained the written informed consent. All subjects suspected to be affected by FD were investigated for GLA gene mutation. Genomic DNA was isolated from whole blood using the EZ1 DNA Blood 350 μl Kit and the EZ1 workstation (Qiagen, Hilden, Germany) following the manufacturer s instructions. The entire GLA gene exons and exon intron boundaries were polymerase chain reaction amplified and sequenced using the ABI 3130xl DNA automated sequencer (Applied Biosystems, Carlsbad, CA) and analyzed with a sequencer 3.0 software (Gene Code Corporation, Applied Biosystems, Carlsbad, CA). Molecular analyses for IDUA and FUCA1 genes were performed as previously described (12). Results Family I A 36-year-old woman, because of her desire to become pregnant, requested a genetic counseling to determine a possible carrier status for fucosidosis, a diagnosis formulated in two cousins of the mother s lineage: the first-born affected child died at the age of 3 (Fig. 1, VII-7.3); the second-born patient underwent a bone marrow transplantation at the age of 5, and now was 11-year old (Fig. 1, VII-7.4). Consanguinity was reported between the proband s parents. The molecular analysis in the two cousins showed the presence of the homozygous nonsense mutation c.244c>t (p.gln82term) in the FUCA1 gene, confirming the reduction of α-fucosidase enzyme. Hence, the molecular analysis of the genotype of the affected patients allowed us to exclude the potential carrier condition for fucosidosis in the proband. However, during a more careful clinical assessment, we observed that the patient had facial features resembling Fabry facies ; in addition, she presented with angiokeratomas on the lips and umbilicus. She also complained tinnitus, occasional gastrointestinal symptoms and severe allergy to nickel. These findings prompted us to perform investigations for FD. The leucocyte GAL-A levels were slightly reduced. Mutation analysis of the GLA gene revealed the genomic mutation c.868a>c (p.met290leu) confirming the diagnosis of FD. This GLA gene mutation was previously reported and characterized, showing a pathogenetic role (13). A comprehensive clinical and instrumental evaluation showed a mild left ventricular hypertrophy (LVH), proteinuria and Gb-3 storages at electron microscopy analysis of the kidney biopsy, signs of previous ischemic episodes at the magnetic resonance imaging. The medical assessment was extended to some other family members, and FD was therefore confirmed in the 57-year-old mother and in the 33- year-old sister (Fig. 1, V-5.3 and VI-6.6). The mother did not show signs and symptoms of FD, whereas the sister, who was under treatment for eating disorders (anorexia bulimia), was recently diagnosed to be affected by multiple sclerosis for visual disturbances. One female subject was simultaneously carrier for fucosidosis and affected by FD (Fig. 1, V-5.2) 282

3 Fabry disease and other LSDs Fig. 1. Pedigree of the Family I. The coexistence of Fabry and fucosidosis was observed. One female subject was simultaneously carrier for fucosidosis and affected by Fabry disease (V-5.2). The great grandfather (Fig. 1, II-2.2, mother s lineage) and the second grandfather of the proband (Fig. 1, II-2.3, father s lineage) were brothers. Family II An 86-year-old woman (Fig. 2, III-3.11) was enrolled into a genetic screening program for FD on the basis of a mild form of LVH. The relative molecular investigations confirmed the diagnosis of Fabry for the heterozygous mutation c.644a>g (p.asp215ser) on the GLA gene, mutation previously characterized with a pathogenetic role (14). Although consanguinity was not reported, proband s parents were native from the same small Italian village. The FD molecular investigations of other family members identified as affected the proband s son in hemizygous status (Fig. 2, IV, 4.10) and two female subjects in heterozygous status (Fig. 2, III-3.3 and III-3.9), who were in good health. Interestingly, during the genetic counseling, the proband reported that her heterozygous FD-affected sister had two children suffering from MPS I Hurler/Scheie (Fig. 2, IV-4.8 and IV-4.9). The molecular analysis of IDUA gene had revealed that the two patients, respectively, 51- and 54-year old, were compound heterozygotes for [c.45_57del12 (p.ser16_ala19del)] + [c g>a (p.val371metfsx43)] currently treated with enzyme replacement therapy (ERT) with α-l-iduronidase [Aldurazyme (BioMarin Pharmaceutical Inc., Novato, CA and Genzyme Corporation, Cambridge, MA)]. Therefore, the 79-year-old sister of the proband was affected by FD and simultaneously carrier for MPS I Hurler/Scheie. Discussion It is well known that the diagnosis of FD can be delayed or even missed because of the variability of the clinical features: signs and symptoms of FD frequently overlap with those of several different pathological conditions. The characteristic acral and joint pain, usually associated with fever, are frequently misdiagnosed as rheumatic fever, fibromyalgia, rheumatoid arthritis, systemic lupus erythematosus or other connective tissue disorders (15). Cryptogenic early-onset stroke and multiple sclerosis can be erroneously diagnosed in patients recognized to be suffering from FD later in life (16). Many FD patients might be missed in nephrology clinics, where they are classified as affected by end-stage kidney disease of unknown origin, or in cardiological clinics, where they are diagnosed as generically affected by cardiomyopathies (4). Since 2001, as a consequence of the availability of the specific ERT, screening for FD has been extended to patients with LVH and to patients with history of early stroke, apparently without other risk factors, and this has increased the diagnosis of FD (17). The patient of Family II was identified after a screening campaign aimed to characterize patients with hypertrophic cardiomyopathy. In the female proband of Family I, the presence of a Fabry facies and angiokeratoma were highly evocative for the disease. Hence, the level of attention for Fabry should be higher, especially during the medical assessment of female patients, because they can present lighter symptoms and signs. 283

4 Zampetti et al. Fig. 2. Pedigree of the Family II with subjects affected by Fabry disease and others by mucopolysaccharidosis I Hurler/Sheie. One female patient, in good health, was simultaneously carrier for MPS I and presented a mutation for Fabry disease. Angiokeratomas are skin lesions also described in other LSDs, including sialidosis, galactosialidosis, adult form of gangliosidosis GM1, aspartylglucosaminuria (AGA) and fucosidosis, this latter disease being known to affect other members of the same family (18). It should be underlined that the enzymatic assay, although diagnostic in male patients, could be unreliable in female Fabry. Therefore, when suspecting the disease in female subjects, the molecular analysis by sequencing the GLA gene should be the gold standard. In the both families, two female subjects were double heterozygotes for Fabry and for fucosidosis or MPS I Hurler/Scheie, respectively. However, while the two autosomal recessive conditions have a low prevalence, respectively, <1:1,000,000 for fucosidosis (<100 patients reported worldwide) (19) and 1:88,000 for MPS I (20), the frequency of the classic X-linked Fabry phenotype has been recently estimated to be at 1 in 40,000 male individuals and that of FD overall (classic and late onset) at 1 in 3100 newborn males screened in Italy (21) or 1 in 1250 Thai newborn males (22). Hence, the frequency of heterozygotes in the population for MPS I is calculated at 1:148 individuals, whereas for FD it should be around the double of the prevalence of the affected males. Thus, it is probably less surprising than previously believed that females who are at risk to be carrier (or are carrier) of a LSD for consanguinity are at the same time heterozygous for FD. Consanguinity was reported in the Family I and not excluded a priori in the Family II, going back to previous unknown generations, because patients came from a small village. In 2001, Guy et al. (23) reported the presence of two LSDs, AGA and Fabry, in a consanguineous family: two subjects first diagnosed as FD and received a second diagnosis of AGA. Consanguinity can increase the risk to be carrier for an autosomal recessive disorder but has no role in the transmission of X-linked disorders like FD. In conclusion, the incidence of Fabry could change in the future thanks to screening programs including female individuals with specific organ morbidities and personal or family history suggestive of this disorder. The risk of coexistence of more than one LSDs in a family pedigree, although rarely reported, should be always considered. Acknowledgements We are grateful to DNA Biobank from Patients Affected by Genetic Diseases (G. Gaslini Institute) Telethon Genetic Biobank Network (Project No. GTB07001A) and Italian Health Department Finanziamento Ricerca Corrente. AMMeC (Associazione Malattie Metaboliche Congenite, Italia) is also kindly acknowledged for its support. References 1. Meikle PJ, Hopwood JJ, Clague AE, Carey WF. Prevalence of lysosomal storage disorders. JAMA 1999: 281: Germain DP. Fabry disease. Orphanet J Rare Dis 2010: 22: Ries M, Gal A. Genotype phenotype correlation in Fabry disease. In: Mehta A, Beck M, Sunder-Plassmann G, eds. Fabry disease: perspectives from 5 years of FOS. Oxford: Oxford PharmaGenesis, 2006 Chapter 34, MacDermot K, Holmes A, Miners AH. Anderson-Fabry disease: clinical manifestations and impact of disease in a cohort of 98 hemizygous males. J Med Genet 2001: 38: Pintos-Morell G, Beck M. Fabry disease in children and the effects of enzyme replacement treatment. Eur J Pediatr 2009: 168: Ramaswami U, Whybra C, Parini R et al. Clinical manifestations of Fabry disease in children: data from the Fabry Outcome Survey. Acta Paediatr 2006: 95:

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