Brain, Vol. 124, No. 7, 1426-1437,
July 2001
© 2001 Oxford University Press
Clinical, biochemical and molecular genetic characteristics of 19 patients with the Sjögren–Larsson syndrome
1 Departments of Paediatric Neurology, 2 Dermatology and 3 Laboratory of Paediatrics and Neurology, University Medical Centre, St Radboud, Nijmegen, Departments of 4 Clinical Biochemistry and 5 Paediatrics, Academic Medical Centre, University of Amsterdam, 6 Department of Neurology, Laurentius Hospital, Roermond, The Netherlands and 7 Division of Metabolic and Endocrine Diseases, University Children's Hospital, Heidelberg, Germany
Correspondence to:
M. A. A. P. Willemsen, Department of Paediatric Neurology, University Medical Centre St Radboud, PO Box 9101, 6500 HB Nijmegen, The Netherlands E-mail: m.willemsen{at}ckskg.azn.nl or R. J. A. Wanders, Departments of Paediatrics and Clinical Chemistry, Academic Medical Centre, University of Amsterdam, PO Box 22700, 1100 DE Amsterdam, The Netherlands E-mail: wanders{at}amc.uva.nl
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Abstract |
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Sjögren–Larsson syndrome (SLS) is an autosomal recessively inherited neurocutaneous disorder caused by a deficiency of the microsomal enzyme fatty aldehyde dehydrogenase (FALDH). We report the clinical characteristics and the results of molecular studies in 19 SLS patients. Patients 1–17 show the classical triad of severe clinical abnormalities including ichthyosis, mental retardation and spasticity. Most patients were born preterm, and all patients exhibit ocular abnormalities and pruritus. Electro-encephalography shows a slow background activity, without other abnormalities. MRI of the brain shows an arrest of myelination, periventricular signal abnormalities of white matter and mild ventricular enlargement. Cerebral 1H-MR spectroscopy reveals a characteristic, abnormal lipid peak. The degree of white matter abnormality in the MRIs and the height of the lipid peak in 1H-MR spectra do not correlate with the severity of the neurological signs. The clinical presentation and the clinical course is strikingly similar in these patients. Patient 18 shows a mild phenotype that essentially contains the same, but less severe, clinical features. Patient 19 exhibits the typical, but very mild, dermatological and ocular abnormalities, without any clinical neurological involvement. The diagnosis of SLS was confirmed by demonstration of the enzyme defect in cultured skin fibroblasts. Furthermore, as might be predicted from the essential role of FALDH in leucotriene B4 (LTB4) metabolism, elevated urinary concentrations of LTB4 and 20-OH-LTB4 were found in all patients studied. Molecular studies of the FALDH gene revealed eight different mutations, including three new ones: a large 26-base pair deletion (21–46del), a missense mutation (80C
T) and an insertion mutation (487–488insA). The vast majority of SLS patients seem to be severely affected independent of their genotype. Sjögren–Larsson syndrome; fatty aldehyde dehydrogenase; leucotriene B4; mutations; genotype-phenotype correlation
ALDH = aldehyde dehydrogenase; FALDH = fatty aldehyde dehydrogenase; LTB4 = leucotriene B4; MRS = magnetic resonance spectroscopy; PCR = polymerase chain reaction; RFLP = restriction fragment length polymorphism; SLS = Sjögren–Larsson syndrome
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Introduction |
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The Sjögren–Larsson syndrome (SLS; McKusick no. 270200) is an autosomal recessive neurocutaneous disorder that was originally recognized in the co-existence of congenital ichthyosis, spastic di- or quadriplegia and mental retardation (Sjögren and Larsson, 1957
; Jagell et al., 1981; Jagell and Heijbel, 1982). Additional clinical findings that have been reported include juvenile macular dystrophy, pruritus and preterm birth (Jagell et al., 1980; Rizzo, 1993; Lacour, 1996; van Domburg et al., 1999; Willemsen et al., 1999a). SLS was originally described in the county of Vasterbotten in northern Sweden. Probably due to founder effects and inbreeding, a high prevalence (8.3 per 100 000) is found in this population while its prevalence over the whole of Sweden is only 0.4 per 100 000. SLS is now known to occur worldwide and, in the absence of clusters of patients reported elsewhere, its prevalence has been estimated as 0.4 per 100 000 or lower. Recently, we have described beneficial effects of the leucotriene B4 (LTB4) synthesis inhibitor zileuton in the treatment of SLS (Willemsen et al., 2000b). No other causative therapeutic options are available at present.
SLS is an inborn error of lipid metabolism caused by a deficiency of the microsomal enzyme fatty aldehyde dehydrogenase (FALDH) (Rizzo et al., 1988; Rizzo and Craft, 1991). This enzyme catalyses the oxidation of medium- and long-chain fatty aldehydes, whether or not derived from fatty alcohols, to the corresponding carboxylic acids (Fig. 1) (Kelson et al., 1997; Verhoeven et al., 1998; Willemsen et al., 2000a). It has been postulated that FALDH deficiency may lead to an accumulation of fatty alcohols or aldehyde-modified macromolecules with structural consequences for cell-membrane integrity, and elevated concentrations of biologically highly active lipids (Rizzo and Craft, 1991; James and Zoeller, 1997; Willemsen et al., 2001).
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In 1994, the FALDH gene was mapped to chromosome 17p11.2 (Pigg et al., 1994
). Its genomic organization and tissue-dependent expression have subsequently been elucidated (Chang and Yoshida, 1997; Rogers et al., 1997). The gene consists of 10 exons and nine introns, and spans ~31 kb (kilobases). The cDNA for FALDH encodes a protein of 485 amino acids. This protein has a hydrophobic carboxyl-terminal amino acid sequence that is assumed to be necessary for microsomal membrane anchoring (Masaki et al., 1994). Alternative splicing of the FALDH gene results in a transcript with an additional sequence (exon 9'). The protein produced from this mRNA is far less abundant, and its function is unknown. Mutation analysis has identified many different mutations in the FALDH gene in SLS patients (De Laurenzi et al., 1996; Rizzo et al., 1997, 1999; Sillén et al., 1997, 1998; Tsukamoto et al., 1997; Willemsen et al., 1999b; Aoki et al., 2000). The extensive report by Rizzo and colleagues shows that SLS is associated with a striking number of different mutations (Rizzo et al., 1999). The available studies that deal with the molecular biology of SLS provide only very limited clinical data. This study aims to outline the clinical signs and symptoms together with the biochemical characteristics and the mutational spectrum of SLS, based on a series of 19 patients from 13 families.
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Patients
Our series consists of 19 patients from 13 families (Table 1
). Patients 1–17 suffer from the classical phenotype with severe involvement of the skin, the CNS and the eyes. Patients 18 and 19 have mild clinical features. Families VII, VIII and IX originate from Turkey, while the other families have their roots in The Netherlands. The parents in Families IV, VIII and IX are known to be consanguineous. The fathers of Patients 1 and 5 are relatives in the second degree. Informed consent was given by Patients 18 and 19, and by parents of Patients 1–17 to take part in this study.
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The degree of spasticity was scored based on the ability to ambulate: the patient is confined to a wheelchair (3); the patient uses a wheelchair in everyday life, but is able to walk a short distance with or without the help of aids (2); the patient has a mild spastic gait or shows involvement of the pyramidal tracts on physical examination (1); there are no signs or symptoms of involvement of the pyramidal tracts (0). Mental performance was measured in 10 patients using standard intelligence tests, and estimated in the other patients based on school performance and communication skills. Mental retardation has been defined as an IQ < 70, and classified according to the WHO criteria (World Health Organization, 1980
). Electro-encephalography was routinely performed in 13 patients using a 32-channel Profile digital EEG-system (Oxford Instruments, Eynsham, UK).
Clinical findings on Patients 2, 4–7, 12–15, 18 and 19 and detailed results of ophthalmologic investigations of Patients 1–9, 12–15, 18 and 19 have been described previously (van Domburg et al., 1999; Willemsen et al., 2000c).
MRI and 1H-MR spectroscopy
Cerebral MRI and 1H-MR spectroscopy (1H-MRS) of the brain have been performed in 16 and 13 patients, respectively, using the methods described previously (van Domburg et al., 1999). All MRI were systematically evaluated and scored. The abnormalities of the cerebral white matter were graded with regard to the intensity of the pathological signal on T2-weighted images (grade 0 = normal white matter; grade 1 = mildly increased signal intensity; grade 2 = severely increased signal intensity). The MR spectra were evaluated with regard to the presence of the characteristic lipid peak (Miyanomae et al., 1995; Mano et al., 1999; van Domburg et al., 1999). For that purpose, MR spectra from the occipital white matter [TE (echo time) 30 ms; TR (repetition time) 3000 ms; volume of interest 20 x 20 x 20 mm] were selected in each patient. In those spectra, we compared the height of the lipid peak with the height of the N-acetylaspartate peak, and graded it as follows: 0 (no lipid peak visible), 1 (lipid peak < N-acetylaspartate peak), or 2 (lipid peak > N-acetylaspartate peak). The spectral signs of the lipid peak, N-acetylaspartate, choline and creatine were not quantified for this study.
Biochemical investigations
The FALDH activity was measured in cultured skin fibroblasts, using octadecanol as the substrate. We used the methods described by Rizzo and colleagues (Rizzo et al., 1988; Rizzo and Craft, 1991), with only minor modifications (Willemsen et al., 1999b).
The concentrations of LTB4, 20-OH-LTB4 and 20-COOH-LTB4 were measured in urine (n = 16) and CSF (n = 8). The LTB4 degradation capacity of fresh polymorphonuclear leucocytes was determined in six patients. The methods used have been described in detail, together with most of the results for SLS patients as a group (Willemsen et al., 2000c).
Mutation analysis
The genomic DNA from peripheral blood cells was isolated for genetic analysis using a diatom matrix as described by Boom and colleagues (Boom et al., 1990).
Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis
For rapid screening of the 943CT mutation, genomic DNA was amplified in a 25 µl PCR reaction mixture containing 10 mM Tris—HCl (pH 8.4 at 25°C), 1.0 mM MgCl2, 50 mM KCl, 0.1 mg/ml bovine serum albumin, 0.2 mM each dNTP (deoxynucleotide triphosphate), 2.5 U Taq polymerase (Promega) and the sense primer 5'-CAG TTC ATC CAC GTG CTC AG-3', and the anti-sense primer 5'-AGA GCC AGA GGC TTT TCA CG-3'. DNA amplification was performed in a PTC–100 thermocycler (M. J. Research Inc., Waltham, USA) programmed as follows: 120 s at 96°C before starting cycling, five cycles of 30 s at 96°C, 30 s at 55°C and 60 s at 72°C followed by 30 cycles of 30 s at 94°C and 60 s at 72°C, with a final extension at 72°C for 10 min. Subsequently, PCR products were digested with MnlI and were analysed by agarose gel (2% w/v) electrophoresis.
Sequence analysis
The FALDH gene was amplified in seven fragments under PCR conditions as described above, using the M13-tagged primers as listed in Table 2. DNA amplification was performed in a PTC–100 thermocycler from M. J. Research Inc. programmed as follows: 60 s at 96°C before starting cycling, five cycles of 30 s at 96°C, 30 s at 55°C and 180 s at 72°C followed by 30 cycles of 30 s at 96°C and 180 s at 72°C, with a final extension at 72°C for 10 min. Sequence analysis of the PCR fragments using BigDye fluorescent labelled M13 primers was performed on an Applied Biosystems 377A automated DNA sequencer (PE Applied Biosystems, Foster City, Calif., USA) following the manufacturer's protocols.
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The clinical features of the patients, the results of MRI, 1H-MRS and EEG, the concentrations of LTB4, 20-OH-LTB4 and 20-COOH-LTB4, the enzyme activities of FALDH and the mutations in the FALDH gene are summarized in Tables 1 and 3
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Clinical features
Patients 1–17
The dermatological manifestations of these patients were strikingly similar. In the first months after birth the cutaneous features were often mild. During the first year of life, however, the skin took on a highly characteristic appearance with brownish yellow discoloration and a markedly wrinkled hyperkeratosis (Fig. 2
). The ichthyosis was generalized, with sparing of the face only. Pruritus was an accompanying disabling feature in all patients, and was present every day. Since the skin disorder was the most prominent sign in early infancy, most patients were first seen by the dermatologist. In some patients the characteristic appearance of ichthyosis was recognized, which led to an early diagnosis of SLS. In others, the diagnosis was made in infancy after emerging neurological signs became apparent.
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Spasticity was found in all 17 patients, it predominantly involved the legs and presented during the first two years of life with gradual worsening. All patients used a wheelchair in everyday life. Patients 1, 5, 6, 12 and 15, however, had maintained the capability to walk a short distance (<25 m) with the use of aids, and Patients 8 and 9 were able to walk up to 100 m unsupported. Joint contractures of the legs had developed in all patients and often necessitated orthopaedic corrections. Speech production, based on pseudobulbar dysarthria in combination with cognitive defects, was affected in all. The patients' mood was strikingly good. All children attended special schools for the mentally handicapped. The mental retardation should be qualified as `mild' to `moderate'. In addition, mental performance was characterized by a marked slowness. Clinical follow-up, parental reports and results at school, as documented by the teaching staff, showed no loss of acquired skills. One or more epileptic seizures occurred in three children, none of them requiring chronic treatment with antiepileptic drugs. The neurological picture varied slightly between the patients, with comparable differences within and between families.
The macular dystrophy was characterized by the presence of retinal crystals, generally referred to as `glistening white dots' (Fig. 3). The number of retinal crystals seemed to increase slightly with age (Willemsen et al., 2000c). Inter-individual differences in the number of dots in patients of the same age group were negligible. Bilateral corrected visual acuity ranged between 0.6 and 0.16. Photophobia was a notable additional finding.
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All EEG recordings (Patients 2, 4–9 and 12–15) showed symmetrical slow background activity with an alpha rhythm frequency of 7–8 Hz. None of the EEGs revealed epileptiform patterns or abnormal changes of amplitude.
Patients 18 and 19
Patient 18, a male, was born after a normal pregnancy and delivery. In the first nine months of life, his skin was unremarkable. Thereafter, a generalized fine scaling of the skin with hyperkeratotic, well-demarcated and yellow-brown coloured plaques developed gradually. Pruritus and photophobia were additional symptoms. Psychomotor development was normal during childhood. Difficulties with walking developed during adolescence, and gradually worsened during the following decades. At the age of 36 years, he was investigated by the neurologist and ophthalmologist. The patient seemed feeble-minded, had a mild spastic gait, and hyperreflexia of the legs and extensor plantar responses. His speech was unremarkable. The ocular abnormalities consisted of subnormal visual acuity, the presence of macular glistening dots and photophobia. The EEG showed a symmetrical background activity with an alpha rhythm frequency of 7–8.5 Hz, without other abnormalities. The patient has two healthy children.
Patient 19, the sister of Patient 18, was born preterm. However, the precise gestational age could only be estimated as `three months too early'. Her skin was reported normal until the age of 9 months, at which time she developed skin symptoms similar to those of her brother but less extensive. The characteristic skin lesions were only found in the main flexor folds of the extremities including the axillae. Occasionally she experienced pruritus. Her intellectual development was normal. There were no motor complaints. Neurological examination revealed no abnormalities. Ophthalmologic examination showed the typical retinopathy and photophobia. Her EEG was normal (alpha rhythm: 10–11 Hz). She has given birth to one healthy child.
The parents and siblings (four brothers and one sister) of Patients 18 and 19 are healthy, and show no neurological or dermatological abnormalities.
MRI and 1H-MRS
The MRI studies showed no gross structural defects. Three abnormal phenomena were observed on T2-weighted images. First, small areas of the subcortical association fibres of the frontal, parietal and occipital convexities, as well as the temporal pole were found to be unmyelinated in all but one patient (Patient 18). Secondly, all patients showed a periventricular zone, extending from frontal to occipital, of hyperintense signal disturbances. Thirdly, mild ventricular enlargement was found in the oldest patients. There were no signs of severe atrophy. 1H-MRS revealed the presence of the characteristic lipid peak in all patients, except Patient 19.
Biochemical characteristics
Urine was determined to be free of protein, erythrocytes and leucocytes. The concentrations of LTB4 and 20-OH-LTB4 were found to be highly elevated in all patients (n = 16), while 20-COOH-LTB4 was absent. This pattern fits with a metabolic block between 20-OH-LTB4 and 20-COOH-LTB4, due to FALDH deficiency. None of the three metabolites were found in healthy controls (detection limit < 5 nmol/mol creatinine). The mean (standard deviation) concentrations of LTB4 and 20-OH-LTB4 in 14 severely affected patients were 29.9 (14.3) and 291.6 (148.2), respectively. The results of Patients 18 and 19 fell within this range.
CSF showed normal values for erythrocytes, leucocytes, total protein, albumin (and Q-albumin), glucose and lactate in all patients (n = 8). Mean (standard deviation) concentration of LTB4 in CSF was 134.5 (22.1) pmol/l, which equals the mean (standard deviation) concentration [114.7 (57.1) pmol/l] in control samples. The concentrations of 20-OH-LTB4 and 20-COOH-LTB4 in CSF were below the detection limit (<15 pmol/l) in SLS patients and controls.
Molecular biology
We found nine different mutations in 13 families (Tables 1 and 3). Three of these mutations [a large 26-bp (base pair) deletion (21–46del), 487–488insA and 80CT] have not been reported before. Homozygosity was found in 10 patients, while eight patients were compound heterozygous. Five mutations were only found in single families. Heterozygosity has been proven in all parents, except in Family XII from which material was not available.
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Clinical aspects
In 1957, Sjögren and Larsson described a series of patients who suffered from a severe disorder with three cardinal features: ichthyosis, mental retardation and spastic di- or quadriplegia. Since then, the syndrome has borne their names. The underlying enzyme deficiency was discovered 31 years later and it took another 6 years before the genetic defect was unravelled. The full clinical spectrum of SLS can now be examined in patients in whom the diagnosis has been proven and the biochemical and genetic basis has been resolved.
The skin disorder in SLS has a characteristic appearance with its prominent generalized hyperkeratosis and remarkable brownish yellow colour. All patients also suffer from severe pruritus. The presence of pruritus in SLS contrasts with other ichthyotic skin disorders, which are generally non-itching. For this reason, pruritus has to be recognized as an important additional symptom that strongly suggests SLS in patients under investigation.
The ocular abnormalities that can be found in SLS seem to be undervalued in the literature. We have recently performed a detailed study on the ophthalmological features in 13 patients and found multiple abnormalities in all patients (Willemsen et al., 2000c). Photophobia, macular dystrophy and decreased visual acuity are the most prominent of the ophthalmological abnormalities. It is important to note that the glistening white dots of the retina can easily be overlooked in young and less cooperative patients who suffer from photophobia. This seems the best explanation for the report of a lower prevalence of macular dystrophy in the literature. The nature of the crystalline deposits in the retina is unclear. It is speculated that they might represent accumulations of long-chain fatty alcohols or fatty aldehydes.
Preterm birth was reported for 12 out of 17 (71%) SLS patients in this series, and seems to be one of the features of the syndrome (Willemsen et al., 1999a). The reason for the preterm birth is unclear. It is hypothesized that it relates to the defective LTB4 degradation in SLS. Despite their preterm birth, none of the patients suffered from perinatal or neonatal complications. For this reason, spasticity in SLS and preterm birth should be interpreted as co-existing features. The spasticity should not be attributed to the prematurity.
The patients' spasticity gradually worsens during the first decades of life, leading to contractures and, in most patients, to wheelchair dependency. The number of retinal crystals seems to increase slightly with age, and cerebral MRI shows a mildly enlarged ventricular system in the oldest patients. These findings might reflect a very slow progressive course of the disease. On the other hand, the mental handicap and the other clinical features of SLS, seem to be non-progressive in all patients.
Patients 18 and 19 exhibit most of the classical features of SLS but they are far less severely affected. Their ichthyosis had the same age of onset and the same appearance as in Patients 1–17, but affects a far less extensive area of the skin. Pruritus is less severe. The characteristic retinopathy was found in both patients. Both patients have a normal intelligence, and only Patient 18 developed a (relatively mild) spastic diplegia from the second decade of life. Barnard and colleagues described two brothers with a marked speech defect, ichthyosis, spasticity and the typical crystalline retinopathy (Barnard et al., 1991). In contrast to the severe spasticity and involvement of the eye and skin, both men were reported to be `responsive and intelligent'. It is not reported whether the diagnosis in this family has been biochemically proven. Nigro and colleagues described three siblings with decreased FALDH activities (in the range of SLS patients), and a mild phenotype (Nigro et al., 1996). All three patients showed mild spasticity, but only two of them had skin abnormalities (demarcated plaques of hyperkeratotic yellow-brown skin). Their speech was normal, and there was no evidence of mental retardation in these patients. Ophthalmological investigations did not indicate any abnormalities. The authors do not report on preterm birth or pruritus. To our knowledge, these three families (Barnard et al., 1991; Nigro et al., 1996; Family XIII reported in this paper) are the only ones with `non-classical' SLS. There are two possible explanations for the rarity of reports on mildly affected SLS patients. First, mild SLS might indeed be extremely rare. The uniformity of the severe clinical features of our Patients 1–17 might favour the assumption that phenotypic variation is uncommon in SLS. The other possibility is, however, that SLS is insufficiently recognized in mildly affected patients. In those cases where the classical features occur together but are mild, it should still be possible to recognize SLS. However, when an isolated `SLS feature' is encountered in the absence of the other two features of the classical triad, it could be very difficult to recognize that the patient is suffering from SLS. As routinely performed laboratory investigations do not offer any biochemical marker for SLS the identification of mildly affected patients is further hampered.
MRI and 1H-MRS
The results of cerebral MRI were abnormal in all patients studied by us, but lack specificity. A clear relationship between the degree of the MRI abnormality and the neurological features could not be demonstrated. 1H-MR spectra revealed a typical lipid peak that was present in all severely affected patients. This sharp lipid peak clearly differs from the lipid signals that are found in other disorders with white matter involvement, and is thought to arise from the accumulation of long-chain fatty alcohols or fatty aldehydes (Miyanomae et al., 1995; Mano et al., 1999; van Domburg et al., 1999). The height of the lipid peak (as compared with the height of the N-acetylaspartate peak) does not seem to predict the severity of the neurological features. Although the findings in only one mildly affected patient do not provide a basis for much speculation, the absence of the peak in Patient 19 might still suggest that its presence is a marker for the severe phenotype of SLS.
FALDH: the enzyme and its substrates
Aldehyde dehydrogenases (ALDHs) are a group of enzymes that catalyse the oxidation of aldehydes to their corresponding carboxylic acids (Yoshida et al., 1998; Perozich et al., 1999). They are widely distributed throughout all species. These enzymes all have their individual subcellular localizations and tissue distributions. Some ALDHs can oxidize only a limited range of substrates, while others have broad substrate specificity. In humans, at least 12 ALDHs have been identified. They are generally referred to as ALDH1 to ALDH9, FALDH or ALDH10, succinic semialdehyde dehydrogenase and methylmalonate semialdehyde dehydrogenase. Phylogenetic studies have demonstrated that the human ALDH3, 7 and 8, and FALDH are closely related. Some regions of the protein are highly conserved in these and other human ALDHs, and in many ALDH families from other species.
The measurement of FALDH activity in leucocytes or cultured skin fibroblasts can easily discriminate between SLS patients and healthy controls when either the C18 aliphatic fatty alcohol (octadecanol) or aldehyde (octadecanal) are used as substrates (Rizzo and Craft, 1991; Rizzo, 1993; van Domburg et al., 1999). In our experience (>40 patients), FALDH residual activities in patients range between 0 and 25% of the mean value in controls, without overlap with the normal range. However, it is assumed that the level of residual activity as measured is higher than the true FALDH activity. One explanation for this phenomenon could be that other aldehyde dehydrogenase isozymes, present in the cytoplasm or in other cell compartments, will, at least under in vitro conditions, also react with octadecanol or octadecanal. This fact has so far hampered the elucidation of the relationship between the genetic defect, the degree of FALDH deficiency and the phenotypic expression. The measurement of the LTB4 degradation capacity of fresh leucocytes might offer a solution to these problems. Briefly, in this method radiolabelled LTB4 is incubated with isolated leucocytes for 15 min, and then the amount of radiolabelled 20-COOH-LTB4 is measured and expressed as the percentage of the initial amount of LTB4. Using this technique, we found that leucocytes of four severely affected patients did not oxidize any (<1%) LTB4 to 20-COOH-LTB4. The leucocytes of the mildly affected patients showed minimal, but unmistakable, residual activity; they were able to convert 3.2% (Patient 18) and 2.7% (Patient 19) of LTB4 to 20-COOH-LTB4. The leucocytes of 10 healthy controls showed activities of 51.4% (SD 5.4%). Although the results of these investigations have to be interpreted with caution because of the limited number of patients that were tested, they seem promising. The measurement of the LTB4 degradation capacity might offer the first opportunity to determine the true residual activity of FALDH in SLS patients. If so, it provides a tool to study the correlation between residual enzyme activity and clinical phenotype.
Free fatty alcohols in plasma, octadecanol and hexadecanol are elevated in SLS patients (Rizzo and Craft, 2000). Technical difficulties in fatty alcohol analyses, however, still hinder their quantification. Moreover, fatty alcohols have also been shown to accumulate in patients suffering from other inborn errors of metabolism (Rizzo et al., 1993). The pathological urinary excretion of LTB4 and 20-OH-LTB4 is a biochemical marker for SLS (Willemsen et al., 2001). All patients have abnormally elevated concentrations of both metabolites, with a normal concentration of 20-COOH-LTB4. This pattern has never been found in other patients. The concentrations of these metabolites in the mildly affected Patients 18 and 19 do not differ from the concentrations in the severely affected patients. Based on these results, we conclude that the analyses of LTB4 and its metabolites in urine can be used as a diagnostic tool for SLS. The excretion pattern is highly characteristic, but the concentrations of the metabolites do not predict the severity of the phenotype.
Biochemical investigations of CSF have so far not revealed any abnormality in SLS patients. Surprisingly, even 20-OH-LTB4 concentrations are normal in CSF. The latter contrasts with the systemic (urinary) values and points to an, as yet unknown, `escape mechanism' of the CNS.
FALDH: the gene
We found nine different mutations in the FALDH gene in 13 families, including three unreported mutations. Overall, missense and deletion mutations seem to occur most often in the FALDH gene of SLS patients (Sillén et al., 1998; Rizzo et al., 1999).
The 943CT mutation leads to the substitution of serine for the highly conserved proline at position 315 in the FALDH protein and is, together with the 1297–1298delGA mutation, held responsible for a large part of all mutations in patients of European origin (De Laurenzi et al., 1997; Rizzo et al., 1997; IJlst et al., 1999). In the patients presented here, the mutations affect 20 out of 36 alleles (56%). The 1297–1298delGA mutation (Rizzo et al., 1997) as well as the 1381–1384delGAAA mutation (Willemsen et al., 1999) are predicted to result in a truncated FALDH that no longer anchors to the microsomal membrane, with consequent loss of enzymatic function. The 798GC mutation has recently been found in a compound heterozygous patient, who also carried the common 943CT mutation (Rizzo et al., 1999). Clinical data for the patient were not given. Expression studies of the 798GC mutation in Chinese hamster ovary cells revealed an enzyme with relatively high (55%) residual activity (Rizzo et al., 1999). Importantly, the authors found that the mutation resulted in an unstable mRNA. Thus, this mutation seems to have a greater effect on the amount of enzyme produced than on the activity of the actual enzyme. Our Patients 1 and 5 are compound heterozygous for the 798GC and the 1297–1298delGA mutation. The presence of the 798GC mutation in these two patients with the classical features of SLS supports its pathogenic significance. Another missense mutation, namely 733GA, was originally regarded as a polymorphism (Sillén et al., 1998), but has subsequently been reported to result in severe reduction of FALDH activity (Rizzo et al., 1999). We found this mutation in an inbred family with two homozygous patients, which confirms its destructive consequences.
Two missense mutations of the FALDH gene, namely 551CT and 943CT, were shown to occur in the siblings with a mild phenotype (Family XIII). Both patients were found to be compound heterozygous for these mutations. Heterozygosity for the 551CT mutation could be proven in the mother and three siblings of the patients, while the father carried the 943CT mutation. The 551CT mutation has previously been described in four probands with SLS (Rizzo et al., 1999). The clinical features in these patients have not been reported. The mutation has been predicted to cause the substitution of threonine (amino acid 184) by methionine (T184M). Threonine 184 is present in human ALDH 1–10 and succinic semialdehyde dehydrogenase, and is part of a well-conserved region of human ALDHs. This threonine is just upstream of two glycine residues (G185 and G190) which participate in NAD+ binding (Liu et al., 1997). The T184M mutation might change the binding of NAD+ and thereby increase the Km for this substrate. We were unable to confirm this hypothesis experimentally, because a 10-fold increase in the concentration of NAD+ did not lead to higher residual activity of the protein (data not shown). Expression of the 551CT mutation into FALDH-deficient Chinese hamster ovary cells resulted in severe loss of FALDH catalytic activity (<1% of wild type) (Rizzo et al., 1999). Based on the actual knowledge about both the 551CT and the 943CT mutations, as summarized above, it seems impossible to explain the mild phenotype of these patients on the basis of their genotype.
The 80CT mutation in exon 1 has not been reported previously. We have found it in one compound heterozygote and one homozygous patient. This mutation leads to the substitution of leucine with proline at position 27. This is the most amino-terminal mutation of any of the previously reported missense mutations. The leucine at position 27 is well conserved among aldehyde dehydrogenases in humans and other species including fungi and bacteria, and is found in the cytosolic and mitochondrial as well as microsomal isozymes (Yoshida et al., 1998; Perozich et al., 1999). Figure 4 illustrates the homology of different aldehyde dehydrogenases and the high conservation of the amino acid leucine at position 27 in FALDH. Insertion mutations are rarely found in SLS, and isolated 1 bp insertions have only been reported in two families (Sillén et al., 1998; Rizzo et al., 1999). The finding of the (new) 487–488insA mutation in exon 4, that causes a frameshift, is remarkable from this point of view. The other new, and large, 26 bp deletion mutation of exon 1 (21–46del) in Patient 4 gives rise to a frameshift from codon 7. Although deletion mutations are quite common in SLS, such gross mutations are rare.
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All reports on the molecular biology of SLS published so far have stated that the patients who were analysed suffered from SLS without giving details of the phenotypes. The phenotype of Patients 1–17 is essentially uniform with regard to the occurring features as well as their course, although slight differences between patients can be found. This clinical homogeneity is in favour of the assumption that there is only slight phenotypic variation within most SLS patients. A wide spectrum of mutations contrasts with a phenotype with consistent severe involvement of the skin, the CNS and the eyes.
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Conclusions |
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On clinical grounds the diagnosis of SLS should be considered in any neonate or infant with a congenital ichthyosis, especially when the child is born preterm. In an infant not yet diagnosed, emerging neurological features should point to SLS. If the diagnosis of SLS comes into consideration, one should look for such additional clinical features as pruritus, retinal glistening dots and photophobia. These additional features are decisive for the clinical diagnosis. Cerebral MRI reveals arrested myelination maturation and periventricular white matter involvement, but these findings lack specificity. The demonstration of the characteristic lipid peak on cerebral 1H-MRS, however, can offer another clue to the diagnosis. In atypical cases, the differential diagnosis might encompass peroxisomal disorders (e.g. Refsum disease), and many other rare syndromes which involve multiple organs (Rizzo, 1993
; Stoll and Eyer, 1999; van Domburg et al., 1999).
The demonstration of elevated concentrations of free fatty alcohols in cultured fibroblasts or in plasma, or the abnormal presence of metabolites of LTB4 degradation in urine, can provide biochemical confirmation of the clinical suspicion of SLS. Definite proof that a patient actually suffers from SLS has to come from measurement of FALDH activity and molecular studies of the FALDH gene. Although some mutations (943CT and 1297–1298delGA) have a high prevalence, in many patients thorough molecular studies will be necessary to reveal the individual genetic defect. Genetic and biochemical studies can also be used for prenatal diagnosis. FALDH activity can be measured in cultured amniocytes or cultured chorionic villus cells. Mutation analysis of a chorionic villus sample is another possibility, especially in those families with a known genetic defect.
From Patients 18 and 19 it can be seen that SLS is not always a severely disabling disorder. We have no clear explanation for the occurrence of a mild phenotype in these patients. One could speculate that unusual dietary habits within the family might have protected the patients from the developing severe signs of SLS, but we did not find arguments for this when discussing this matter with the patients. Moreover, it is known that a high proportion of the fatty alcohols in the human body is not ingested, but rather produced by endogenous lipogenesis. Compensating mechanisms have to be looked for in a beneficial genetic background. These patients may help us to reveal the mechanisms that beneficially influence the signs and symptoms of SLS. Moreover, they should motivate us to look for SLS in less severely affected patients with unexplained ichthyosis, spasticity or ocular abnormalities.
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The authors wish to thank L. Folkers, Laurentius Hospital, Roermond, The Netherlands; M. S. van der Knaap, Department of Child Neurology, Free University Hospital, Amsterdam, The Netherlands; J. R. M. Cruysberg, Institute of Ophthalmology, M. van der Graaf, Department of Radiology, A. Heerschap, Department of Radiology, M. Lutt, Department of Paediatric Neurology, J. W. Pasman, Department of Clinical Neurophysiology, R. C. A. Sengers, Department of Paediatrics, H. O. M. Thijssen, Department of Radiology, and A. Verrips, Department of Paediatric Neurology, University Medical Centre St Radboud, Nijmegen, The Netherlands, for the examination of the patients, the evaluation of the MRIs, MRSs and EEGs, and their contributions to the preparation of this manuscript. We also thank the (referring) clinicians who offered us the opportunity to include all patients in this study. This study was supported in part by a grant from the Deutsche Forschungsgemeinschaft to E.M. (Ma1314/2–3).
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Received December 4, 2000. Revised February 15, 2001. Accepted February 22, 2001.
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