Clinical phenotype of lathosterolosis

� 2007 Wiley-Liss, Inc. American Journal of Medical Genetics Part A 143A:2371–2381 (2007)

Clinical Phenotype of Lathosterolosis

Massimiliano Rossi,1 Maria D’Armiento,2 Ida Parisi,1 Paola Ferrari,3 Christine M. Hall,4

Mariarosaria Cervasio,2 Francesco Rivasi,5 Fiorella Balli,3 Raffaella Vecchione,2

Gaetano Corso,6,7 Generoso Andria,1 and Giancarlo Parenti1*1Dipartimento di Pediatria, Federico II University, Naples, Italy

2Dipartimento di Scienze Biomorfologiche e Funzionali, Sezione di Anatomia Patologica, Federico II University, Naples, Italy3Dipartimento Materno Infantile, University of Modena and Reggio Emilia, Modena, Italy

4Department of Radiology, Great Ormond Street Hospital for Children, London, UK5Dipartimento di Scienze Morfologiche e Medico Legali, Sezione di Anatomia, Istologia e Citologia Patologica,

University of Modena and Reggio Emilia, Modena, Italy6Dipartimento di Biochimica e Biotecnologie Mediche, Federico II University, Naples, Italy

7Dipartimento di Scienze Biomediche, University of Foggia, Foggia, Italy

Received 19 December 2006; Accepted 27 May 2007

Lathosterolosis (LS) is a defect of cholesterol biosynthesisdue to the deficiency of the enzyme sterol-C5-desaturase.Only two patients have been described to date, bothpresenting with multiple malformations, mental retardation,and liver involvement. In addition in one of them patho-logical examination revealed mucolipidosis-like inclusionson optic microscopy analysis, and peculiar lysosomal lamel-lar bodies on electron microscopy analysis. This study isfocused on a better characterization of the clinical phenotypeof LS. We describe a further case in a fetus, sibling of the firstpatient reported, presenting with neural tube defect, cranio-facial and limb anomalies, and prenatal liver involvement.The fetal phenotype suggests the possible occurrence ofsignificant intrafamilial variability in LS, and expands thephenotypic spectrum of the disease. Histological examina-tion of autopsy samples from the fetus and skin fibroblastsfrom the living sibling suggested that the mucolipidosis-like

picture previously reported is not a constant feature of LS,being possibly associated with the most severe biochemicaldefects, but confirmed the ultrastructural finding of lamellarinclusions. The LS phenotype appears to be characterizedby the distinctive association of a recognizable pattern ofcongenital anomalies, involving axial and appendicularskeleton, liver, central nervous and urogenital systems, andlysosomal storage. This condition partially overlaps withother defects of sterol metabolism, suggesting intriguingpathogenic links among these conditions.� 2007 Wiley-Liss, Inc.

Key words: lathosterolosis; Smith–Lemli–Opitz syndrome;neural tube defect; butterfly vertebra; Niemann–Pick type C;storage; polydactyly

How to cite this article: Rossi M, D’Armiento M, Parisi I, Ferrari P, Hall CM, Cervasio M, Rivasi F, Balli F,Vecchione R, Corso G, Andria G, Parenti G. 2007. Clinical phenotype of lathosterolosis.

Am J Med Genet Part A 143A:2371–2381.

INTRODUCTION

Defects of cholesterol biosynthesis (DCB) are agroup of inherited metabolic diseases associatedwithmultiple congenital anomalies andmental retardation(MCA/MR). Smith–Lemli–Opitz syndrome (SLOS,OMIM: #270400), due to the deficiency of the enzyme7-dehydrocholesterol reductase (DHCR7), is themostfrequent and best-characterized example of DCB[Kelley and Hennekam, 2000; Porter, 2003]. SLOSpatients present with typical facial dysmorphisms,limb anomalies, incomplete development of malegenitalia, and malformations possibly involvingall organs and systems. Lathosterolosis (LS,OMIM: #607330) is an additional example of thisgroup of metabolic syndromes [Parnes et al., 1990;

Brunetti-Pierri et al., 2002; Krakowiak et al., 2003;Rossi et al., 2005]. This disorder is due to thedeficiency of sterol-C5-desaturase (SC5DL), theenzyme immediately upstream DHCR7, which cata-lyzes the transformation of lathosterol into 7-dehy-drocholesterol. Inheritance is autosomal recessive. LSis very rare: in fact after the characterizationof the firstcase [Brunetti-Pierri et al., 2002], only one other LSpatient has been diagnosed postmortem [Krakowiak

*Correspondence to: Prof. Giancarlo Parenti, Department of Pediatrics,Federico II University, Via Sergio Pansini 5, 80131 Naples, Italy.E-mail: [email protected]

DOI 10.1002/ajmg.a.31929

et al., 2003]. Interestingly, the latter case showedmassive mucolipidosis-like inclusions [Parnes et al.,1990; Krakowiak et al., 2003], a feature not evident onhistological examination of a liver biopsy performedin the first case reported [Rossi et al., 2005]. The aim ofthis study is a better characterization of the clinicalphenotype of LS. Firstly, we describe a new LS case ina fetus sibling of the first patient reported, expandingthe clinical spectrum of the disease. We also reportupdated clinical and morphological informationabout the first patient diagnosed [Brunetti-Pierriet al., 2002; Rossi et al., 2005]. We then compare thephenotypic features observed in LS with thosereported in SLOS, and other defects of sterolmetabolism. Finally, a few hypotheses correlatingthe observed pattern of abnormalities to possibleunderlying pathogenic mechanisms are discussed.

CLINICAL REPORTS

Patient 1

Clinical features. The first sibling of the livinggirl with LS [Brunetti-Pierri et al., 2002; Rossi et al.,2005], was a historical fetal case, aborted at 21 weeksgestation, after a routine ultrasound scan revealedmultiple malformations. Neither maternal diabetes,nor teratogenic exposure was reported. Around con-ceiving the mother was on adequate folate supple-mentation. Clinical features were reviewed after adiagnosis of LS was made in the living sister. Onmacroscopic examination, weight and crown-rumplength appeared to be appropriate for gestationalage (290 g and 21 cm, respectively). Postmortemfindings included MCA such as Type II Arnold Chiarimalformation, microcephaly, postaxial hexadactylyof upper and lower limbs, bilateral clubfeet andlumbosacral meningocele. No malformations werereported in other organs. Unfortunately neitherphotos nor radiographs were available. Chromo-some analysis showed a normal female karyotype.A diagnosis of LS was suspected on the basis of thepattern of limb and craniofacial anomalies. Molec-ular analysis of the SC5DL gene, performed aspreviously described [Brunetti-Pierri et al., 2002] inDNA samples obtained from stored pathologicalspecimens, revealed the same two mutations foundin the living sister (p.R29Q and p.G211D), thusconfirming the diagnosis.Histological features. Histological examina-

tion of fetal samples from thymus, spleen, pancreas,liver, kidneys, adrenals, uterus, ovaries, heart, lungs,spinal cord, and brain was carried out by opticmicroscopy using the following stains: hematoxylin-eosin; special histochemical stains like PAS and PASafter diastase digestion, Hale’s dialyzed colloidal ironfor acid MPS; immunostaining for HepPar1 in liverhistology. In none of the histological specimensexamined, were cells with storage or foamy material

noted. In particular, liver tissue showed normalportal tracts, and marked extramedullary hemato-poiesis leading to atrophy of hepatocytic laminae(Fig. 1). Hemosiderinic pigment within periportalhepatocytes and Kupffer cells were also present(Fig. 1 detail). Hypoxic changes were noted in cere-bral cortex and white matter, likely due to abortionprocedures. No significant histological changes werenoted in the other organs.

Patient 2

Clinical features. This girl with LS, presentedwith developmental delay, craniofacial anomalies,postaxial polydactyly of the left foot, bilateralminimal soft-tissue syndactyly between the 2nd and4th toes and, on the left side, soft tissue syndactylybetween the 5th toe and the extra digit, horseshoekidneys, bilobate gallbladder and progressive intra-hepatic cholestasis [Brunetti-Pierri et al., 2002; Rossiet al., 2005]. She was reviewed at the age of 7 years.During follow-up, her facial gestalt had significantlychanged. In the neonatal period the girl showed astriking microcephaly, with receding forehead, eye-lid ptosis, prominent nose with bulbous nasal tip andmicrognathia with protruding upper lip (Fig. 2A).In the following years, physical examination reveal-ed microcephaly with bitemporal narrowing, epi-canthic folds, eyelid ptosis, a small nose withanteverted nares, a small chin, puffy cheeks, and along philtrum (Fig. 2B,C). At 6 years of age, smallbilateral lens opacities became evident on ophthal-mological examinations, which subsequently evolv-ed in total cataracts of the right eye, requiringsurgery. The patient had severe cholestasis with liverfibrosis and persistently elevated serum levels oftransaminases, g-glutamyl transferase, alkaline phos-

FIG. 1. Patient 1: liver histology showing normal portal tracts with markedextramedullary hematopoiesis leading to atrophy of hepatocytic laminae.Hemosiderinic pigment within periportal hepatocytes and Kupffer cells wasalso noted (detail; H&E, 200�; detail: HepPar, 400�).

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American Journal of Medical Genetics Part A: DOI 10.1002/ajmg.a

phatase, total and direct bilirubin and ammonia, asdescribed in details in a previous report [Rossi et al.,2005]. In the last reviews, liver disease had furtherprogressed to severe liver failure: portal hyper-tensionwasnotedon abdominalDoppler ultrasoundat 7 years of age, and liver transplant was consideredas a life-saving procedure. In spite of multi-vitaminsupplementation, low levels of vitamins A and Ewere repeatedly detected, as well as abnormalclotting tests. The girl experienced two pathologicalfractures and DEXA scan performed at 7.5 yearsof age, revealed severe generalized osteoporosis(Z-score: �4.9).

At 6.5 years of age, plasma sterol profile by gas-chromatography-mass spectrometry showed normallevels of cholesterol (155 mg/dl), persistently high

levels of lathosterol (17 mg/dl), and detectable levelsof methylsterols, such as in the previously performeddeterminations [Brunetti-Pierri et al., 2002]: thesemetabolites were identified as the plant sterolssitosterol (0.7 mg/dl), campesterol (0.5 mg/dl), andstigmasterol (0.2 mg/dl; total plant sterols: 0.8% oftotal sterols detected; Pianese and Corso, unpub-lishedwork). This biochemical featurewas not notedin over 20 SLOS patients diagnosed in our Center(Pianese and Corso, unpublished work), includingtwo patients with severe cholestasis [Rossi et al.,2005]. Patient 2’s diet was free and, in particular, wasnot vegetarian. She had frequent diarrhea aftertasting cow milk and some derivatives, and showeda striking fishy body odor after eating fish, which shetended to avoid spontaneously: urine trimethyl-

FIG. 2. Patient 2: evolution of facial appearance at different ages. A: Neonatal period: note microcephaly, receding forehead, prominent nose with bulbous nasal tipand micrognathia with protruding upper lip. B: At 2 years of age: note a significant change in facial gestalt compared with the neonatal age. C: At 7 years of age: notemicrocephaly, cataract of the right eye, epicanthic folds, eyelid ptosis, a small nose with anteverted nares, a small chin, and a long philtrum.

CLINICAL PHENOTYPE OF LATHOSTEROLOSIS 2373


amine analysis by Protonic Nuclear Magnetic Reso-nance was negative [Maschke et al., 1997], and noclear explanation was found for this phenomenon.

Blood smears revealed the presence of abnormallyshaped red cells with anisopoikilocytosis (MCV: 89.3fl; normal values: 82–98; RDW: 18.4%; normal values11–14), acanthocytes, schistocytes, and large plate-lets (mean platelet volume: 12.4 fl; normal range:9.1–12.3); vacuolated monocytes were also noted(Fig. 3). Full blood count repeatedly showed normalhemoglobin levels (range: 12.2–13.2 g/dl) and lowto normal platelet count (range: 121–219� 103/ml;normal range: 150–400� 103/ml).Radiological features. A skeletal survey, per-

formed at 11 months of age, showed sagittal cleftingof the eighth thoracic vertebra (‘‘butterfly’’ shaped;Fig. 4A), as previously mentioned [Brunetti-Pierriet al., 2002]. In addition, the left foot showedpostaxial polydactyly characterized by ossificationof a proximal phalanx, and an abnormal widening ofthe 5th metatarsal representing an equivalent ofduplication. Radiologically, postaxial polydactylywas evident also in the right foot, as shown by theabnormally wide 5th metatarsal, with no phalangealossification (Fig. 4B).

A comparison was made between the limb radio-logical anomalies noted in Patient 2 and those foundin six unrelated SLOS cases diagnosed at theDepartment of Pediatrics, Federico II University ofNaples with available skeletal surveys. These patientsshowed significant similarities, such as toe syndac-tyly (6/6), and a polydactyly pattern including eitherpostaxial (4/6) or interdigital (involving the 3rd–4thweb spaces; 1/6) extra-digits (Fig. 4C).Histological features. Skin fibroblasts from

Patient 2 were expanded in DMEM supplementedwith 10% fetal bovine serum. Cells were harvested atT0 and after 3 and 7 days of culture in delipidated

medium, as previously described [Brunetti-Pierriet al., 2002]. Subsequently, fibroblasts were fixed in2.5% glutaraldehyde in 0.1 M cacodylate buffer,postfixed in 1% osmium tetroxide and dehydratedthrough graded alcohols for Epon embedding;sections were stained doubly with uranyl acetateand lead citrate, and examined by a Zeiss EM-109electron microscope.

These cells were compared with skin fibroblastsfrom two non-related SLOS patients diagnosed at theDepartment of Pediatrics Federico II University, andone control, cultured in the same conditions.

The LS cells showed lamellar lysosomal inclusionbodies at T0 (Fig. 5A). Fibroblasts from two SLOSpatients also showed almost indistinguishable lamel-lar inclusions (Fig. 5C). In both cases the lamellarvacuoles appeared to be progressively degradedwithin the lysosomes after three and, more exten-sively, after 7 days of culture in delipidated medium(Fig. 5B). Control cells did not show any sign ofstorage (Fig. 5D).

DISCUSSION

Cholesterol plays a crucial role in several biologicalpathways including steroid hormone biosynthesis,embryonic development, and the definition ofcellular membrane functional microdomains inter-acting with various signaling pathways [Kelleyand Herman, 2001; Gondre-Lewis et al., 2006]. As aconsequence of this biological pleiotropy, a disrup-tion in cholesterol biosynthesis can lead to a widerange of congenital anomalies with significantclinical variability [Kelley and Herman, 2001]. Thisis well exemplified by desmosterolosis, a DCB due tothe deficiency of 3-b-hydroxysterol-D24-reductase(OMIM: #602398): the two patients described to dateshowed striking differences in their clinical pictures,including micro- and macrocephaly and very differ-ent patterns of bone mineralization [FitzPatrick et al.,1998; Andersson et al., 2002]. The definition of theclinical phenotypes of DCB is crucial to increase therate of diagnoses and to correlate specific patterns ofanomalies to the underlying pathogenic mecha-nisms. LS represents a further example of a rareDCB showing a significant phenotypic variabilityeven within the same family, as demonstrated by thenew case here described. Nevertheless, based on thevery few patients reported to date, the phenotype ofLS appears to be characterized by a recognizablepattern of multiple malformations, involving partic-ularly axial and appendicular skeleton, liver, centralnervous and urogenital systems, partially overlap-ping with SLOS (Table I).

Facial Features

The neonatal craniofacial phenotype of our Patient2 is characterized by a marked microcephaly (Fig. 2),and shows remarkable similarity to the non-related

FIG. 3. Patient 2: hematological features: mp: macroplatelets; aca, acantho-cytes; sch, schistocytes; vm, vacuolated monocytes (detail; May–Grunwald–Giemsa).

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described case [Parnes et al., 1990]. Figure 2 clearlyshows that a significant change in her facial appea-rance occurred in the following years, with a gestaltprogressively becoming more similar to the SLOSfacies.

Axial Skeleton

Patient 1 presented with lumbar meningocele.Neural tube defects (NTD) are a group of congenitalanomalies ranging from anencephaly, resulting fromfailure of fusion of the cranial neural tube, to spinabifida, resulting from a failure of the vertebrallaminae to fuse to complete the neural arch, withan overall incidence of approximately 1/1,000[Merbs, 2004; Mitchell, 2005]. These malformationshave been reported either in MCA/MR syndromesdue to chromosomal abnormalities (e.g., trisomies13, 18, and 21), single gene defects (e.g., Meckel–Gruber syndrome, OMIM: #249000), and teratogenicexposures (e.g., maternal diabetes), or as isolateddefects [Mitchell, 2005]. The vast majority of isolatedcases are multi-factorial and periconceptional folatesupplementation to the mother has been proved toreduce significantly the incidence of these devel-opmental defects [Mitchell, 2005]. It is difficult to

evaluate whether the occurrence of NTD in thepatient here described is specifically related to theunderlying metabolic disease as a consequence ofthe disruption in cholesterol metabolism. A fewobservations demonstrate that severely reducedsterol levels in mice can lead to a disruption inneural tube closure. Mice lacking squalene synthase,an enzyme involved upstream in cholesterol biosyn-thesis, whose deficiency has not been reported inassociation with human phenotypes until now, doshow severe NTD [Tozawa et al., 1999]. NTD havebeen described also in mice homozygous or hetero-zygous formutations of the apoBgene, which plays akey role in cholesterol transport [Homanics et al.,1995; Huang et al., 1995]. Nonetheless, to the best ofour knowledge NTD have never been reported todate in biochemically confirmed patients with DCB,including very severely affected SLOS patients withvery low plasma sterol levels [Kelley and Hennekam,2000; Porter, 2003]. Lumbar meningomyelocele hasbeen described in a case with a clinical diagnosis ofCHILD syndrome (OMIM: #308050) [Hebert et al.,1987], but it might be worth emphasizing that,although CHILD syndrome is currently classifiedamong the DCB, neither low cholesterol norabnormally high levels of cholesterol precursors

FIG. 4. Radiological features found in lathosterolosis Patient 2 and comparison with Smith–Lemli–Opitz syndrome. A: Patient 2, aged 11 months: detail of spineradiograph, showing the sagittal clefting of the eighth thoracic vertebra, ‘‘butterfly’’ shaped (white arrow). B: Patient 2, aged 11 months: detail of feet showing bilateralpostaxial polydactyly characterized on the left side by an ossified proximal phalanx and abnormal widening of the 5thmetatarsal representing an attempt at duplication,and, on the right side, abnormally wide 5th metatarsal, with no duplicated phalangeal ossification.C: Unrelated SLOS patient, aged 9 months: detail of feet. In the rightfoot there is postaxial polydactyly with fusion at the base of the 5th and 6th metatarsals. The left foot shows soft tissue syndactyly and interdigital polydactyly, with asmall extra metatarsal and a proximal phalanx in the 3rd and 4th web space.



are usually found in the affected patients [Waterham,2006]. Furthermore, studies evaluating apoE andapoB genotypes in humans with NTD have failed tofind a significant association [Volcik et al., 2002], andto the best of our knowledge NTD have not beenreported in patients affected by abetalipoproteine-mia (OMIM: #200100) and hypobetalipoproteinemia(OMIM: þ107730) or in their offspring. Furtherstudies are finally necessary to evaluate whether ornot abnormally high levels of lathosterol can beassociated with a particular affinity for inducingNTD.

Patient 2 presented with an eighth thoracic‘‘butterfly’’ vertebra. Sagittal clefting is a rare ana-tomical defect which has been reported as a part ofmultiple malformation syndromes, such as Alagillesyndrome (OMIM: #118450), or as an isolatedanomaly [Sonel et al., 2001]. This is the result of adevelopmental error involving the cartilaginousprecursor of the vertebral centrum, associated witha failure of the notochord to recede [Kjaer et al., 1994;Merbs, 2004]. It has been demonstrated that DCB are

associated with a functional disruption in differentsteps of the Sonic Hedgehog (Shh) pathway, which isessential for embryogenesis and, particularly, fornormal midline development [Porter, 2003; Henne-kam, 2005]. In particular, Shh is expressed in thefloor plate and the notochord, is required for thespecification of the ventral spinal cord [Gofflot et al.,1999; Lupo et al., 2006], and represents one ofthe major factors from the notochord and floor platepromoting and controlling the formation of thesclerotome, which is the primary origin of the axialskeleton [Kornak and Mundlos, 2003]. Therefore, itmight be possible to hypothesize that an impairmentof the Shh pathway, secondary to the defectivecholesterol biosynthesis, contributes to the observedvertebral midline defect.

Appendicular Skeleton

The two LS patients here described, presented withpostaxial polydactyly associated, in Patient 2, withtoe syndactyly. Both these features were also shown

FIG. 5. Ultrastructural features found in lathosterolosis Patient 2 and comparison with Smith–Lemli–Opitz syndrome. A: Skin fibroblasts from Patient 2, at baseline,16,000�. Note the lamellar inclusions (white arrows). B: Skin fibroblasts from Patient 2, after 7 days of culture in delipidated medium, 40,000�. The lamellar vacuolesappear to be progressively degraded within the lysosomes (white arrows). C: Skin fibroblasts from an unrelated Smith–Lemli–Opitz patient at baseline, 13,000�,showing almost indistinguishable lamellar inclusion bodies (white arrows). D: Skin fibroblasts from a control, after 7 days of culture in delipidated medium, 12,000�,showing no sign of storage.

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by the patient described by Parnes et al. [1990],showing hexadactyly with complete metatarsus andphalanges in the left foot, and fused 5th and 6thmetatarsal bones on the right, associated with

bilateral 2–3 toe syndactyly. The LS mouse modelshows postaxial polydactyly and, in addition, inter-digital defects with bifurcation of the middlephalanges of the fourth rays [Krakowiak et al.,

TABLE I. Phenotype of Lathosterolosis, and Comparison With the Smith–Lemli–Opitz Syndrome

Clinical features

LS

SLOS (frequency %, if known)Patients (no. of reports) LS mouse model

A: Congenital anomaliesGender 2 Female, 1 male NA NAGrowth delay þ (2/3) þ þIntrauterine growth retardation þ (1/3) þ þ (16)Postnatal growth retardation þ (2/2) NAc þ (82–88)Facial dysmorphisms þ (3/3) þ þ (100)Microcephaly þ (3/3) � þ (80–84)Prominent metopic suture þ (1/3) � þEyelid ptosis þ (2/3) NA þ (59–70)Downslanting palpebral fissures þ (1/3) NA þCataract þ (2/2) � þ (12–22)Corneal clouding/microcorneae þ (1/3) � þShort nose þ (2/3) þ þAnteverted nares þ (1/3) NA þ (69–78)Micrognathia þ (2/3) þ þ (67)Downturned mouth þ (2/3) NA þHigh arched palate þ (2/3) � þ (29)Cleft palate � (0/3) þ þ (37–47)Gingival hypertrophy þ (2/3) � þ (37)Appendicular skeleton anomalies þ (3/3) þ þPostaxial polydactyly þ (3/3) þ þ (48–49)Interdigital defects (bifurcatedphalanges, complete polydactyly)

� (0/3) þ þ

Toe syndactyly þ (2/3) � þ (90–97)Clubfeet þ (1/3) NA þ (27)Axial skeleton anomalies þ (2/3) � þVertebral clefting þ (1/3) � �Neural tube defect þ (1/3) � �Brain structural anomalies þ (2/3) � þ (21–37)Type II Arnold Chiari malformation þ (1/3) NA �Hydrocephalus ‘‘ex vacuo,’’demyelination, dystrophic calcification

þ (1/3) � þ

Developmental delay þ (2/2) NA þ (92–95)Seizures þ (1/2) � þ (<5)

Hearing loss/abnormal auditory evokedpotentials

þ (1/2b) NA þ (10)

Renal malformations þ (1/3) � þ (29–43)Male genital anomalies þ (1/1) � þ (65–91)Liver involvementa þ (3/3) þ þ (2.5–16)Platelet/red cell abnormalities þ (1/1) NA �References Our patients; [Brunetti-Pierri

et al., 2002; Krakowiak et al.,2003; Parnes et al., 1990;

Rossi et al., 2005]

[Krakowiak et al., 2003] Our patients; [Kelley andHennekam 2000; Rossi et al.,

2005; Ryan et al., 1998]

StorageLS

SLOSPatients (no. of reports) LS mouse modelB: Signs of storage

Lamellar inclusions (EM) þ (2/2) þ þMucolipidosis-like inclusions (OM) þ (1/3) � �Vacuolated monocytes (blood film, OM) þ (1/1) NA �Fundus oculi cherry red spot � (0/2) NA �Urine mucopolysaccharides andoligosaccharides

� (0/2) NA �

References Our patients; [Krakowiak et al.,2003; Parnes et al., 1990]

[Krakowiak et al., 2003] Our patients; [Wassif et al., 2002]

IA: congenital anomalies; IB: signs of storage; LS: lathosterolosis; SLOS: Smith–Lemli–Opitz syndrome; no.: number.þ, Reported; �, not reported; OM, optic microscopy; EM, electron microscopy; NA, not applicable.aRanging from mild prenatal involvement to postnatal severe progressive intrahepatic cholestasis.bThe patient reported by Parnes et al. [1990] had abnormal brainstem auditory evoked potentials; our Patient 2 had only transient conductive hearing loss.cEarly lethality.



2003]. We compared the limb anomalies observed inLS with those found in SLOS, reviewing the radio-logical features noted in six unrelated SLOS patients,and the findings previously reported in the literature.It is very well known that SLOS patients generallypresent with soft tissue toe syndactyly, frequentlyinvolving the 2nd and 3rd toe with a distinctive ‘‘Y-shape,’’ and can have postaxial polydactyly [Ryanet al., 1998; Kelley and Hennekam, 2000]: in addition,we observed the presence of the less commoninterdigital polydactyly (Fig. 4C). Postaxial polydac-tyly has been reported also in some of the other DCB,such as Greenberg dysplasia (OMIM #215140) andPelger–Huet homozygosity syndrome (OMIM#169400), both of them caused by mutations in theLBR gene [Oosterwijk et al., 2003], and X-linkeddominant chondrodysplasia punctata (CDPX2,OMIM #302960), due to the deficiency of the enzymeD8-D7-sterol isomerase emopamil-binding protein[Kelley and Herman, 2001]. Therefore, the spectrumof limb anomalies, including both postaxial andinterdigital polydactyly possibly involving the fourlimbs, and syndactyly of toes, appears to be asignificant common pattern of abnormalities of theappendicular skeleton in DCB.

Both Sonic and Indian hedgehog are expressed inlimbs during embryogenesis, playing a central role inregulating the anteroposterior patterning of the limbbud, and the chondrocyte proliferation and hyper-trophy, respectively [Kornak and Mundlos, 2003].Therefore, a functional disruption in the hedgehogpathway might well explain the pathogenesis of theDCB common pattern of limb anomalies.

Chondrodysplasia punctata was not noted onskeletal surveys performed in infancy in two LSpatients [Patient 2; Parnes et al., 1990]. Although thisis a major feature of some of the DCB such as CDPX2,Greenberg dysplasia, and CHILD syndrome, to thebest of our knowledge it has been reported in onlyone biochemically confirmed SLOS case, who hadalso a chromosomal translocation [Kelley andHennekam, 2000], and has not been associated withother DCB, such as desmosterolosis, and mevalonicaciduria (OMIM: #610377).

Other Congenital Anomalies

Both our Patient 2 and the patient described byParnes et al. [1990] developed bilateral cataracts.Cataract has been reported in some of the other DCBsuch as SLOS, mevalonic aciduria, and CDPX2 and inanimal models of DCB such as rats treated with theDHCR7 inhibitor AY9944 [Sakuragawa, 1976]. It hasbeen hypothesized that the accumulation of abnor-mal cholesterol precursors might significantly affectlens metabolism and that cataract might be consid-ered as a storage disease-like manifestation at least inthe SLOS [Cenedella, 1996; Elias et al., 1997]. On theother hand, the recent characterization of a rat model

of hereditary cataract due to the deficiency ofanother enzyme involved in cholesterol biosynthesisnamed lanosterol synthase, and characterized byreduced cholesterol levels without significant pre-cursors accumulations within the lens, has suggestedthat cholesterol deficiency might itself contribute tocataractogenesis too [Mori et al., 2006]. Other ocularfindings associated with LS are eyelid ptosis, down-slanting palpebral fissures, microcornea and cornealclouding, the latter possibly representing anotherexample of storage disease-like manifestations. As inthe SLOS, the development of male genitalia can beaffected in LS [Parnes et al., 1990], while femalegenitalia appeared to be normal in both cases heredescribed. The two patients described postnatallyhad very severe developmental delay [Patient 2;Parnes et al., 1990], in one case associated withseizures [Parnes et al., 1990]. The neurologicalstructural abnormalities reported include Type IIArnold Chiari malformation, brain atrophy(hydrocephalus ‘‘ex vacuo’’), demyelination anddystrophic calcification: further clinical reports arerequired to understand whether there is a definitepattern of central nervous system malformations inthis condition.

It has been recently reported that LS and SLOS micemodels show a marked decrease in the numberof secretory granules, and an increase of morpho-logically aberrant granules in exocrine and endo-crine glands [Gondre-Lewis et al., 2006]. This seemsto be the result of the abnormally high levels ofcholesterol precursors, which significantly affect cellmembrane functional microdomains playing a keyrole in granule budding. Further studies are requiredto clarify whether these functional and morpholog-ical anomalies are detectable also in LS and SLOSpatients, and their clinical relevance.

Signs of Storage

An intriguing finding, reported in the non-relatedLS case previously described, was a widespreadstorage of mucopolysaccharides and lipids withinhistiocytes and the white matter, strikingly sparingthe neurons, and evident as massive inclusions onoptic microscopy. These findings were initiallymisleading, and mucolipidoses were considered inthe differential diagnosis [Parnes et al., 1990]. Nosigns of storage were visible on histological exami-nation by optic microscopy of either a liver biopsyfrom Patient 2 [Rossi et al., 2005], or various tissuesfrom our Patient 1, or tissues from LS mouse model[Krakowiak et al., 2003], suggesting that the wide-spread mucolipidosis-like picture previouslydescribed does not appear to be a constant featureof LS. Since the patient described by Parnes et al.[1990] showed a biochemical defect more severethan our Patient 2 [Krakowiak et al., 2003], it might bepossible to hypothesize that the mucolipidosis-like

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picture is associated only with the most severeenzymatic deficiency. Pre- or peri-natal lethalitymight also have contributed to the lack of overtlysosomal storage observed in the LS mouse model[Krakowiak et al., 2003], and in our Patient 1.

On the other hand, vacuolated monocytes werenoted on blood film examination of Patient 2, afeature previously not investigated [Parnes et al.,1990]. Moreover, electron microscopy examinationof fibroblasts from Patient 2 revealed the presence oflysosomal vacuoles with concentric lamellar inclu-sion, as previously described in the unrelated patientand in the LS mouse model [Parnes et al., 1990;Krakowiak et al., 2003]. We found similar lamellarinclusions also in fibroblasts from SLOS patients(Fig. 5C) according to one previous observation[Wassif et al., 2002]. These findings share strikingmorphological similarities with the typical lysosomalvacuoles visible in fibroblasts from patients affectedby Niemann–Pick type C disease (NPC, OMIM:#257220, #607625) [Patterson et al., 2001; Wassifet al., 2002; Krakowiak et al., 2003], a lysosomalstorage disorder due to mutations of the NPC1 orNPC2 genes, and characterized by a defect incholesterol trafficking. This morphological overlaphas been partially explained. In the SLOS, it has beenshown that 7-dehydrocholesterol impairs low den-sity lipoproteins (LDL) intracellular trafficking anddegradation, probably interacting with the NPC1protein through its sterol sensitive domain. Thisresults in an accumulation of LDL-derived unesteri-fied cholesterol, which can be demonstrated as anincreased filipin staining [Wassif et al., 2002]. Asimilar effect has been postulated also for lathosterolin LS [Krakowiak et al., 2003]. Interestingly, the sameinclusions can be found also in either a rat modeltreated with the DHCR7 inhibitor AY9944 [Sakur-agawa, 1976], or in a cell model treated with class 2amphiphiles, a various group of compounds disrup-ting intracellular lipid trafficking possibly through aninhibition of the NPC1 protein function [Lange et al.,2000; Wassif et al., 2002]. In fact, the lamellar bodiesmight be considered the morphological expressionof a disruption in intracellular cholesterol trafficking,which is the primary defect in NPC, and a secondarydefect in LS and SLOS.

Similar inclusions have been described also indermal cells from a case with CHILD syndrome[Hashimoto et al., 1998], supporting the hypothesisthat disruptions in various steps of cholesterolbiosynthesis might be associated with an abnormalintracellular lipid trafficking.

The NPC clinical phenotype shares some similar-ities with LS, such as developmental delay andintrahepatic cholestatic liver disease with high levelsof gamma-glutamyl transferase [Yerushalmi et al.,2002; Rossi et al., 2005]. Although the presence oflysosomal lamellar inclusions on electron micro-scopy examination and increased filipin staining in

skin fibroblasts is generally considered pathogno-monic of NPC [Patterson et al., 2001], these morpho-logical findings can be demonstrated also in LS[Krakowiak et al., 2003]. The lack of multiple mal-formations, and the presence of specific neurologicalsigns such as ataxia or the typical vertical supra-nuclear ophthalmoplegia can usually clearly differ-entiate NPC from LS and, in selected cases, tissuesterol profiling or molecular analysis of NPC1 andNPC2 genes can be performed for a definite differ-ential diagnosis.

Liver Involvement

The liver involvement in Patient 2 has beenpreviously extensively described [Rossi et al., 2005].Concerning Patient 1, some of the hepatic histolog-ical abnormalities noted have been previouslyreported among non-specific signs of prenatal liverdisease in other DBC, such as desmosterolosis andGreenberg dysplasia [Clayton, 2003]. Our datasuggest that liver involvement in LS may range fromprenatal hepatopathy topostnatal severeprogressiveintrahepatic cholestasis.

The detectable levels of plant sterols found in ourPatient 2 can be tentatively explained by a reducedliver clearance secondary to the severe cholestasis:anyway further reports are necessary to evaluatewhether or not this is a constant feature of LS.

Hematology

Patient 2 showed large platelets, inconstantly lowplatelet count, and abnormally shaped red cells(Fig. 3). Unfortunately no information is available onplatelet size and erythrocyte shape of the patientreported by Parnes et al. [1990], and further reportsare necessary to assess the frequency of theseanomalies in LS. Anyway several factors mightcontribute to the red cells shape abnormalities notedin our patient, such as malabsorption secondary tothe severe hepatic disease, and abnormal erythrocytemembrane fluidity secondary to the abnormal sterolmetabolism. In this respect, it might be interesting tonotice that acanthocytosis is also found in disordersof cholesterol transport, such as abetalipoproteine-mia and hypobetalipoproteinemia [Stevenson andHardie, 2001]. To the best of our knowledge plateletabnormalities have not been reported as a featureof DCB. Large platelets possibly associated withthrombocytopenia, and abnormally shaped red cells(stomatocytes) have been described in Sitosterole-mia (OMIM: #210250), a condition characterized byan impaired transport into the bile and intestinallumen of plant sterols and cholesterol, abnormallyhigh plasma levels of these compounds, and acomplex clinical phenotype including hemolysis,xanthomas, arthritis, accelerated atherosclerosis, andnormal psychomotor development. In this respect, it



might be worth emphasizing that the levels of plantsterols found in our LS Patient 2, even if not detectedin over 20 SLOS cases diagnosed in our Center, arestill within the range reported in the literature for thegeneral normal population [Kuksis et al., 1976] and,on the basis of the information available to date,there is no evidence suggesting links to the plateletanomalies observed.

CONCLUSION

In conclusion, we report a further case of LS,contributing to the delineation of the clinicalphenotype of this condition, which is characterizedby the distinctive association of a recognizablepattern of congenital anomalies and signs oflysosomal storage. Patients presenting with a varia-ble association of malformations involving axial andappendicular skeleton, central nervous and urogen-ital systems, liver dysfunction and developmentaldelay, should undergo plasma or tissue sterolanalysis, or molecular analysis of the SC5DL gene.The clinical phenotype of LS shows a significantoverlap with other DCB such as the SLOS, and sharesalso some similarities with defects of cholesteroltrafficking (NPC), suggesting intriguing pathogeniclinks among these conditions.

ACKNOWLEDGMENTS

We are grateful to Prof. Antonio Dello Russo andDr. Pierluigi Pianese, Dipartimento di Biochimicae Biotecnologie Mediche, Federico II University,Naples, Italy, for their important contribution in theinterpretation of chemical analyses, and to Dr.Antonio Risitano, Divisione di Ematologia, FedericoII University, and Dr Roberta Migliorati, S.C. PediatriaOncologica, Ospedale Pausilipon, Naples, Italy, fortheir knowledgeable clinical opinions.

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