Zellweger spectrum disorders: clinical manifestations in patients surviving into adulthood

The ZSDs have long been considered lethal in infancy or early childhood, based on the original description of Zellweger syndrome (Bowen et al 1964). In this retrospective study, we describe a large cohort of patients with a ZSD and show that the natural history is highly variable with a distinct subgroup surviving well into adulthood. The phenotypic spectrum is therefore much wider, with implications for counseling of patients and their families.

We attempted to identify clinical, biochemical, genetic and/or MRI characteristics typical for this subgroup with long survival. We divided the patients in this cohort into two categories based on the degree of communication. Patients in group 1 (patients 1–12) were able to communicate with structured grammatical speech and patients in group 2 (patients 13–19) did not achieve structured speech. The correlation between the phenotype and genotype, at least with respect to the mild PEX1 c.2528G>A and PEX26 c.292C>T mutation in its homozygous form versus the more severe compound heterozygosity (PEX1 c.2097insT+PEX1 c.2528G > A), which was described previously (Rosewich et al 2005) (Bader et al 2000), was also seen in our study.

In previous case reports, age at first symptoms varied from 3 to 12 years and age at diagnosis varied from 10 to 51 years (Rosewich et al 2005) (Mignarri et al 2012) (Matsui et al 2013) (Raas-Rothschild et al 2002) (Régal et al 2010). The average age at diagnosis in our cohort was at a much earlier age of 5.2 years. Hence, we had the possibility to monitor the clinical and biochemical spectrum over a relatively long period of time.

Hearing impairment, an important characteristic of ZSD patients (Moser et al 1995), was not reported in some of the mild adult patients described in previous reports (Régal et al 2010) (Sevin et al 2011) (Steinberg et al 2009), but all patients in our cohort had impaired hearing and vision. The predominant neurological symptom in the adult patients is a gait disorder, caused by combinations of a cerebellar syndrome, pyramidal tract dysfunction and peripheral neuropathy. Unexpectedly, we observed a high prevalence of signs of peripheral neuropathy in adolescence/adulthood (11/19), especially in group 2 (6/7), while none of these patients presented these symptoms in childhood. The majority of patients was diagnosed with a peripheral neuropathy on clinical grounds. Confirmatory nerve conduction studies, however, were only performed in two patients, which is a limitation of our study. As reported previously (England et al 2005), the combination of signs and symptoms have a relatively good accuracy for diagnosing a peripheral neuropathy. It is noteworthy that two patients developed a demyelinating peripheral neuropathy. This contrasts with another peroxisomal disease, X-linked adrenoleukodystrophy, in which axonal rather than a demyelinating peripheral neuropathy is usually present (van Geel et al 1996) (Engelen et al 2011) (Chaudhry et al 1996).

Previous MRI studies showed progressive cerebral demyelination in peroxisome biogenesis disorder patients with a mild phenotype, mostly in the cerebellum, brainstem, posterior limb of the internal capsule and posterior cerebral white matter (Barth et al 2004). In this study, the major MRI abnormalities (i.e., white matter hyperintensities) were found in the central white matter of both cerebellar hemispheres and/or areas surrounding the dentate nuclei on T2-weighted images (9/16). These lesions can also be found in patients with D-bifunctional protein deficiency, an isolated peroxisomal β-oxidation defect, with prolonged survival (i.e., >7.5 years) (Ferdinandusse et al 2006). Normal MRI was found in five patients. Symptomatic leukoencephalopathy was present in seven patients (2, 5–6, 9, 12, 17–18) and silent leukoencephalopathy in two (3, 8). Overall, individuals in both groups presented similar findings on MRI ranging from normal findings to leukoencephalopathy.

Liver dysfunction is a common feature in ZSD patients. However, in this cohort only four patients (in group 2) had liver cirrhosis/fibrosis. The prevalence of hyperoxaluria (2/14) and hyperglycolic aciduria (3/13) was much lower than previously reported in ZSD patients older than 1 year (van Woerden et al 2006). Only patient 18 suffered from nephro- and urolithiasis.

In our cohort 12 patients were clinically stable in recent years and seven showed a progressive disease course. The disease progression becomes apparent in adolescence (age 12–16 years), with gait disturbance being the most prominent symptom (in 7/7).

Seventeen of the 19 patients had a typical ZSD biochemical phenotype in blood at the time at diagnosis. At latest follow-up we observed normal blood levels of several peroxisomal biomarkers in patients (Table 4). Normal levels of some parameters have already been reported by others (Sevin et al 2011) (Ebberink et al 2010). Importantly, our study is the first to show a decline in the levels of these metabolites with age and in some patients even a complete normalization. In particular the levels of DHCA, THCA, and pipecolic acid were found to decline during life and they eventually normalized. In some patients we noted a decrease in these parameters as well as in liver enzymes. This suggests that improved liver function might play a role in the decrease of these metabolites, as they are predominantly synthesized in the liver (Ferdinandusse and Houten 2006). However, there were also patients with normal liver function and elevated levels of abnormal peroxisomal metabolites in childhood. In these patients we also observed a decline in these peroxisomal parameters, meaning that this decline cannot be entirely attributed to normalization of liver functions. The decline in DHCA and THCA levels may also be caused by a decreased synthesis of bile acids with increasing age (Einarsson et al 1985). Furthermore, we observed strong fluctuation of several parameters in the majority of the patients. The concentration of peroxisomal metabolites can fluctuate between normal and abnormal (Fig. 1). Overall, the diagnosis ZSD would be missed in two patients (2 and 11) at last follow-up and in patient 4 at first analysis based on the C26:0, bile acid levels, pristanic- and phytanic acid concentrations in plasma.

Our data suggest that a ZSD cannot be excluded by biochemical testing in plasma alone, and that in some individuals (i.e., patient 2, 4, and 11) a complete analysis in skin fibroblasts (including culturing fibroblasts at 40 °C (Ebberink et al 2012)) is indicated if clinical suspicion is high. Skin fibroblast examination is also necessary to discriminate between a ZSD or a single-enzyme deficiency, which is diagnosed in at least 15 % of the individuals presenting with a ZSD clinical phenotype (Steinberg et al 2003). Recently, D-bifunctional protein-deficiency has been identified by whole-exome sequencing in seven adults without detectable biochemical abnormalities in blood (Pierce et al 2010) (McMillan et al 2012) (Lines et al 2014). Because of considerable overlap between Usher syndrome and milder ZSD phenotypes, i.e., the combination of deafness and retinitis pigmentosa, individuals suspected to have Usher syndrome should be screened for peroxisomal dysfunction (Raas-Rothschild et al 2002).

Efforts to correlate biochemical, cellular, and molecular characteristics of ZSD patients to their clinical outcome, mainly in terms of survival, have provided valuable information. Gootjes et al suggested that DHAPAT and C26:0 β-oxidation activity are predictors of survival in patients with a ZSD. However, these measurements in cultured skin fibroblasts could only be used to roughly predict survival of <1 year or >5 years (Gootjes et al 2002). Despite these associations, prediction of prognosis remains challenging and is influenced by many other variables (e.g., quality of supportive care). We evaluated how the parameters of peroxisomal dysfunction relate to disease severity and long-term progression. We noticed higher levels of abnormal peroxisomal metabolites (e.g., DHCA, THCA, and C26:0) in group 2 during childhood, but there were no differences in adulthood. Concentrations of pipecolic acid were higher in those patients with a progressive disease course. Patients in group 2 had higher levels of C26:0 in plasma than patients in group 1 throughout life (Fig. 1d). Ferdinandusse et al reported that C27-bile acid intermediates (DHCA/THCA) induce apoptosis, decrease cell viability and are more toxic than C24-bile acids (Ferdinandusse et al 2009). It is noteworthy that the most severely affected patient (18) had extremely high plasma levels of DHCA and THCA ranging from 50 to 130 μmol/l in the first year of life. The patients with a less severe phenotype presented with DHCA and THCA levels of approximately 10 and 2 μmol/l, respectively. However, on the basis of the present study, we were unable to identify a correlation between the concentration of abnormal peroxisomal metabolites, skin fibroblast data and the severity of the clinical phenotype or progression of the disease. The metabolites measured in plasma probably do not reflect the level of accumulation in organs. This suggests that the wide spectrum of clinical presentations may be related to varying amounts of residual peroxisomal enzymatic activities in target tissue. Further studies are therefore required to determine the precise role of the peroxisomal biomarkers in the pathogenesis of ZSDs. The recently constructed PEX1 mouse model (Hiebler et al 2014) may be a valid model to study these relations.

In summary, we describe the natural history of a cohort of ZSD patients who reached adulthood and represent a distinct subgroup within the ZSDs. A high percentage of patients show pyramidal symptoms with or without peripheral neuropathy. Some patients with prolonged survival present an insidiously progressive disease course, despite normalization of biomarkers for peroxisomal disease measured in plasma and erythrocytes. This study emphasizes that ZSDs should no longer be considered solely as a paediatric disease, but rather as a slowly progressive disease with patients surviving into adulthood. This study is important for the interpretation of future therapeutic trials and for those involved in the clinical care of adult patients.