Fabry Nephropathy: Indications for Screening and Guidance for Diagnosis and Treatment by the European Renal Best Practice

Wim Terryn; Pierre Cochat; Roseline Froissart; Alberto Ortiz; Yves Pirson; Bruce Poppe; Andreas Serra; Wim Van Biesen; Raymond Vanholder; Christoph Wanner


Nephrol Dial Transplant. 2013;28(3):505-517. 

In This Article

Screening Methods

2.1 We recommend using enzyme activity measurement for α-Gal A as a primary tool in males, followed by confirmation with mutation analysis when positive. (Ungraded statement)

2.2 We suggest using mutation analysis as a primary tool for screening in females. (Ungraded statement)

Measurement of α-Gal A activity in leucocytes using the fluorogenic substrate 4-methylumbelliferyl-α-D-galactopyranoside is the gold standard for FD in men, with a sensitivity and specificity of nearly 100%. Recently, a dried blood spot test (DBS) using filter paper has been proposed as an alternative to the leucocyte tests.[27] These samples are easy to transport and are stable at room temperature for many days, making it a most convenient screening tool in men, as it is a very sensitive tool with a negative predictive value reaching 100%.

In women, due to skewed X inactivation, enzyme activity measurement has a low sensitivity, as one in three women with FD have normal or nearly normal α-Gal A activity.[15] For this reason, enzymatic tests are less suitable and systematic genetic testing should be encouraged in females with unexplained CKD and manifestations suggestive for FD. As genetic testing is expensive (150–1000 Euro and more per test), a thorough anamnesis, family history and clinical investigation could help to select female CKD patients in whom testing is cost-effective (Figure 1).

Figure 1.

Flowchart for screening for FD in CKD patients.

In FD, gene mutation analysis is a way of confirming diagnosis in male patients, subsequent to enzyme activity measurement. A fresh blood sample can be collected for this purpose, or polymerase chain reaction amplification can be performed on DNA eluted directly from the filter paper used for the DBS α-Gal A measurement.[28]

GLA gene mutations causing FD include single base changes leading to missense or nonsense mutations, or affecting consensus splice sites, small deletions or insertions, but also large gene rearrangements in <5% of the patients. Correlations between a specific mutation, i.e. the genotype, and the severity of the disease, the phenotype, are poor in FD. In a few cases, however, knowledge on the underlying mutation can provide information concerning prognosis and therapy and help the clinician in counselling. Some mutations are frequently associated with an attenuated phenotype, such as the mutation p.N215S, which gives a cardiac phenotype with only LVH.[29] These mutations are associated with a residual enzyme function.[30] A significant proportion of the mutations in men are, however, associated with a very low or absent enzyme function and the classic phenotype.

The GLA gene should be sequenced. As most of the mutations are 'private', i.e. unique to a family, it is always possible to completely identify a previously undetected mutation, and regular updates of such new mutations are available (https://www.hgmd.cf.ac.uk/ac/index.php). The pathogenicity of novel gene alterations such as missense or intronic mutations must always be evaluated. However, in females with normal biochemical tests, it may be difficult to confirm or exclude the diagnosis of FD when a variant of unknown significance is present.

In a suggestive clinical situation, most sequence alterations in exonic regions are pathogenic with very few exceptions. One example of such inert exonic polymorphism is the p. 'D313Y' substitution (G to T at cDNA nucleotide 937); while the plasma enzyme activity towards the artificial substrate is significantly reduced, additional studies demonstrated high residual lysosomal enzyme activity and no pathologic excretion of urinary Gb-3. As a result, the p.D313Y substitution is now generally considered to be a so-called pseudo-deficiency.

If one finds a novel sequence variation in an intronic region or a novel missense mutation that is not known to be a polymorphism present in the general population, several methods allow non-invasive diagnostic analysis to establish whether it is disease causing. First, it should be checked whether these sequence variations exist in the normal population (using electronic databases or an own control population). The second step is to check male relatives of the index case who are carriers of the sequence variation for α- A activity. If the sequence variation is present in some of them, despite a normal α-Gal A activity and absence of clinical manifestations of FD, the sequence variation can be considered to be a polymorphism. If it coincides with a deficient α-Gal A in one or more of the male relatives, the possibility of a disease causing mutation is realistic, and in this case, a work-up of all carriers for the presence of (subclinical) FD disease manifestations should be considered.

Besides enzyme activity measurement and mutation analysis, detection of the accumulating substances (glycosphingolipids) has been studied as a tool for diagnosis. Globotriaosylceramide (Gb-3) is the most important glycosphingolipid, and it should be measured in urine rather than in plasma. Urinary Gb-3 can be a useful diagnostic tool in female heterozygotes with classical FD as it is increased in 95% of them. However, the proportion is much lower in heterozygotes with variant forms. It can also be used in males as a surrogate marker to evaluate the response to ERT.[31] Mass spectrometric profiling of Gb-3 isoforms may also help to identify heterozygotes.[32]

In plasma, deacylated Gb-3 (globotriaosylsphingosine, 'lysoGb-3') has been shown to have a better correlation with FD. It is elevated 200–400 times in males with classical disease, from an early phase in the disease, but its levels can remain low in asymptomatic females or in the 'cardiac variant' p.N215S in males.[33–37] The examination of the urinary sediment with phase-contrast microscopy under polarized light shows tubular cells containing particles with birefringent Maltese Crosses, having a lamellated appearance with protrusions, and consisting of accumulated Gb-3. In the hands of Selvarajah et al.,[38] this was a highly sensitive and specific tool for screening of FD, but its accuracy is strongly operator-dependent and therefore, it is probably an unrealistic option for large-scale screening studies.