- Email: [email protected]
Rett syndrome: New observations
Rett Syndrome: New Observations Sakkubai Naidu, MBBS, Subroto Chatterjee, PhD, Mary Murphy, MBBCh, Sumio Uematsu, MD, Michel Phillapart, MD and Hugo Moser, MD
Two elderly females with Rett syndrome are reported with evidence of a slowly progressive central and distal peripheral nervous system involvement. Thermography in 4 girls confirmed distal hypothermia of the extremities in a glove and stocking distribution. Unilateral sympathectomy during surgery for scoliosis in one of them resulted in increased warmth and physical growth of the foot and nails, compared to the uninjured side. This suggests increased sympathetic tone as the probable cause of distal hypothermia, vasomotor instability and dystrophy of the feel in this disorder. In an attempt to identify a marker, girls with clinically classical Rett syndrome had plasma and urinary cell evaluation for an unusual glycolipid. A blind study using a small number of patients failed to prove absolute specificity and additional studies are required to evaluate its validity as a marker for Rett syndrome. Key words: Elderly Rett, telethermography, sympathetic neuropathy, glycolipid abnormality. Naidu S, Chatterjee S, Murphy M, Uematsu S, Phillapart M, Moser H. Rett syndrome: new observations. Brain Dev 1987; 9:525-8
Dr. Andreas Rett merits lasting recognition for his astute clinical observation, which drew attention to this phenotype , common to females alone. We present two of the oldest cases of Rett syndrome on record and the possible occurrence of a progressi~ axonopathy, involving the distal sympathetic system as evidenced by skin temperature measurements with computerized telethermographic units [1]. We would also like to report our preliminary observation on the presence of an abnormal glycolipid in 9/10 patients with clinically classical Rett syndrome. To evaluate the specificity of the glycolipid in Rett syndrome, a blinded study was conducted using patients with seizures, autism and normals as controls. Older Patients and Telethennography Studies Methods and results In an effort to estimate the number of Rett syndrome patients who were institutionalized, we visited five residential facilities. We identified six patients confOrming to
From Kennedy Institute for Handicapped Children, Baltimore (SN, MM, HM); Johns Hopkins Hospital, Baltimore (SN, SC, SU, HM) ; and UCLA, Neuropsychiatric Institute, Center for Health Science (MP). Correspondence address: Sakkubai Naidu, MBBS, Kennedy Institute, Johns Hopkins Hospital, Department of Neurology and Pediatrics, 707N. Broadway, Baltimore, MD 21205. USA.
the classical Rett syndrome phenotype. They ranged in age from 24 to 42 years. Two of them had been known to us previously. Observations in these two older Rett syndrome patients are tabulated in Table 1. Due to the recognition of early onset of cold extremities with vascular instability in this disorder thermographic studies were performed in four girls aged 5-14 year-old patients with Rett syndrome. The details of the thermographic techniques used in this study have been described elsewhere [1]. Thermography confirmed the clinical observation of distal coldness in all extremities but most severe in the lower extremities. Of particular interest are the findings in a 14-year-old girl who has an inadvertent unilateral left sympathectomy during surgery for scoliosis (Fig 2). The left leg was 3°C and the left foot was O.7°C warmer than the right. There was also a disproportion in size; the left foot was 1.5 cm longer than the right, and the width of the nail on the left big toe exceeded that on the right by2mm.
Discussion Skin temperature is a function of sympathetic vasomotor control and reflects a disturbance in peripheral nerve function. Using telethermography, direct measurement of skin temperature records segments corresponding to the known distribution of sensory and sympathetic nerves, and demarcates these areas [5]. That sympathectomy resulted in increased warmth and vascularity is not surpris-
Table 1 Neurological
Behavior
Other
1-42 yrs
Intense gaze, hyperventilation, bruxism, breath holding, hand wringing
Selective distal wasting lower legs, absent DTR's at knees, feet small and plantar flexed
Scoliosis, pneumonia, phenobarbital and dilantin, seizure free many years
11-42 yrs
Intense gaze, hyperventilation, hand wringing
Contractures and rigidity, predominant distal wasting lower legs, absent DTR's both LE.
Severe seborrhea, chronic mastitis, edentulous, no scoliosis, phenobarbital, tegretol and tranxene
ing. The increase in the size of the foot and nails compared to the opposite side 5 years later, however, raises certain important questions as to the etiopathology of the distal w'asting and cold extremities with vascular instability in this disorder_ Increased sympathetic tone needs to be considered. The preliminary observations by Percy et al [6] and Jellinger et al [7] suggest an axonopathy with increased filaments in the axons and increased clusters of small unmyelinated fibers. This axonopathy may be related to the postulated distal dysfunction of the sympathetic nervous system. There is no clear evidence
of other systemic or central sympathetic abnormalities in this disorder. The elderly Rett syndrome patients were both nonambulatory. Deep tendon reflexes were absent in the legs, but present in the upper extremities. There was severe selective wasting of the muscles of the lower leg with foot deformities. Unfortunately, detailed neurophysiologic studies could not be performed in these older patients. It has been postulated that Rett syndrome may be static after the first decade of life, Progressive increase in rigidity over a seven year period in case II and the
A
GbOse3 Car ·" :1:::',.· ~
.
~:
r;
'Yo
~ I.
~ ~,
o
, 2 34 '5 6
Fig 1 Thin layer chromatography of plasma glycosphingolipids. Suitable aliquots of freshly drawn plasma were extracted with organic solvents (2/ and glycosphingo/ipids isolated employing silicic acid column chromatography [3]. Suitable aliquots of the total glycosphingolipid fraction representing one ml of plasma were separated on HPTLC plates. Authentic standards of neutral g[ycosphingolipids and gang/iosides were co-chromato-graphed and developed simultaneously. The developing solvent was chloroform-metha'lo[-water (containing 0.02% CaCI 2 ) 60 :40: 9. Following development, the chromatogram was sprayed with aniline-diphenylamifle reagent and photographed. A. lane ], human kidney, CbOse3Cer; lane 2; CM 1 ganglioside; lane 3 and 4 CD 2 and CD10 standards; lane 5, 6 plasma glycosphingo-/ipids from two non identical twins EB. unaffected f lane 5) and KB, affected (lalle 6) . B. lane 1, human kidney CbOse3Cer; lane 2, patient (KO) clinically affected but negative, 3, 4, 5, 6 affected patient, KB, A.D., N.c., N.c., lane 7, normal human plasma.
526
Brain & De velopment, Vol 9, No 5, 1987
Fig 2 Thermogram of legs and feet of a 14-yr-old girl with Rett syndrome who had a unilateral left lumbar sympathectomy at 9 yrs of age. The temperature below the right knee was reduced; by O. 7°ein the midleg region and by 3°eat the right foot. The right foot is smaller than the left. This finding suggests that the distal wasting in Rett syndrome patients may be a reflection of increased sympathetic tone.
distal wasting in both older patients raises concerns of a slowly progressive central and peripheral nervous system disorder. The progressive loss of deep tendon reflexes in both the older cases may be a reflection of the peripheral nerve changes observed by Percy et al [6], Jellinger et al [7] and be relevant to our thermographic findings. Detailed serial neurophysiologic and neuropathological studies will help to clarify the nature of these changes. Glycolipid Studies Methods and results Ten patients between 3 1/2 to 21 years of age with the classical clinical presentation of Rett syndrome were randomly selected for the glycospingolipid (GSL) assay. Three were black, six were white, and one of East Indian origin. Four were fasting samples. Five of the ten were on various anticonvulsants for seizures. Ten cc of blood was collected in an EDT A tube and plasma was immediately obtained and kept refrigerated at 4°C until the time of assay. Random urine samples of at least 44 cc were obtained and spun to obtain cells. Total lipids were extracted from plasma organic solvents as described previously [2]. The total lipid extracts were dried in N2 atmosphere and fractionated by silicic acid column chromatography [3]. The GSL fraction were subjected to alkali-catalyzed methanolysis to remove all contaminating phospholipids and dialyzed. The total G'SL fraction were separated by high per-
formance thin layer chromatography employing chloroform-methanol-water (containing .02% CaCh) (60:40:9, V/V). One set of chromatoplates were stained with aniline-diphenylamine reagent to detect the GSLs and photographed. The second set of chromatoplates were dried and exposed to iodine vapor. Gel area co~respond ing to standard GSL and a disialo ganglioside (GD 2) were scraped, eluted with organic solvents and dried in N2 atmosphere. The HPTLC purified samples were subjected to acid-catalyzed methanolysis. Total sphingosine was extracted from the methanolysates by extraction with solvents and quantified [4]. Nine out of 10 children demonstrated a novel glycolipid that migrated in the vicinity of GD2 (disialoganglioside) but was devoid of sialic acid. This substance was present whether the patients were fasting or post prandial and whether or not anticonvulsants were being taken. The negative result occurred in a 17 -year-old girl with typical clinical features, but who was born at 32 weeks gestation and never had the normal early development so characteristic of the Rett's child. A chromatograph of the plasma samples is shown in Fig 1. Since our observations on the new glycolipid were initially promising, a blind study was then conducted with 10 Rett syndrome patients compared to equal numbers of normal girls, and girls with various types of seizure disorders on anti-convulsants. The 6 patients with autism were all boys, in whom its incidence is higher. We noted an increased association (5/10) between the abnormal glycolipid and Rett syndrome girls. One normal girl, one girl with seizures and three of the autistic boys were also positive. It should also be mentioned that difference in testing conditions did occur in the blinded study; namely the test samples analyzed in some of the younger children was small in volume and may have contributed to reduced recovery of the GSL in all groups. Also, all samples were frozen at _80°C for various lengths of time until all of the patients had been accumulated. Under these conditions the association of this glycolipid with Rett syndrome did not reach statistical significance, and therefore the specificity of this assay to Rett syndrome is not clear. Discussion While the possible association between Rett syndrome and an unusual glycolipid is a provocative finding, the data available at this time do not permit final appraisal of its diagnostic Significance or pathogenetic role. Studies are in progress to evaluate a larger group of Rett syndrome and control patients as well as to define the structure of this compound. Once this is accomplished, we aim to raise an antibody to it, and then to utilize a radioimmunoassay to evaluate its potential as a marker for Rett syndrome.
Naidu et al: Rett sy ndrome 527
ACKNOWLEDGMENTS Thi& study was supported in part by an Institutional Research Grant from Johns Hopkins University School of Medicine.
REFERENCES 1. Uematsu S, Long DM. Thermography in chronic pain. In: Uematsu S, ed. Medical thermography, theory and clinical applications. Los Angeles: Brentwood, 1976,52-68. 2. Fo1ch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lip ides from animal tissues. ] Bioi Chern, 1957;226:497-506.
528 Brain & Development, Vol 9, No 5, 1987
3. Sweeley CC, Walker B. Determination of carbohydrates in glycolipides and gangliosides by gas chromatography. Anal Chern 1964;36:1461-6. 4. Lauter CJ, Trams EG. A spectrophotometric determination of sphingosine. ] Lipid Res 1962;3: 136-8. 5. Brelsford KL, Uematsu S. Thermographic presentation of cutaneous sensory and vasomotor activity in the injured peripheral nerve. ] Neurosurg 1986;62:711-5. 6. Percy AK, Zoghbi H, Riccardi VM. Rett Syndrome: initial experience with an emerging entity. Brain Dev (Tokyo) 1985;7:300-4. 7. Jellinger K, Seitelberger F. Neuropathology of Rett syndrome. Am] Med Genet 1986;24 (suppl 1):259-88.
Two elderly females with Rett syndrome are reported with evidence of a slowly progressive central and distal peripheral nervous system involvement. Thermography in 4 girls confirmed distal hypothermia of the extremities in a glove and stocking distribution. Unilateral sympathectomy during surgery for scoliosis in one of them resulted in increased warmth and physical growth of the foot and nails, compared to the uninjured side. This suggests increased sympathetic tone as the probable cause of distal hypothermia, vasomotor instability and dystrophy of the feel in this disorder. In an attempt to identify a marker, girls with clinically classical Rett syndrome had plasma and urinary cell evaluation for an unusual glycolipid. A blind study using a small number of patients failed to prove absolute specificity and additional studies are required to evaluate its validity as a marker for Rett syndrome. Key words: Elderly Rett, telethermography, sympathetic neuropathy, glycolipid abnormality. Naidu S, Chatterjee S, Murphy M, Uematsu S, Phillapart M, Moser H. Rett syndrome: new observations. Brain Dev 1987; 9:525-8
Dr. Andreas Rett merits lasting recognition for his astute clinical observation, which drew attention to this phenotype , common to females alone. We present two of the oldest cases of Rett syndrome on record and the possible occurrence of a progressi~ axonopathy, involving the distal sympathetic system as evidenced by skin temperature measurements with computerized telethermographic units [1]. We would also like to report our preliminary observation on the presence of an abnormal glycolipid in 9/10 patients with clinically classical Rett syndrome. To evaluate the specificity of the glycolipid in Rett syndrome, a blinded study was conducted using patients with seizures, autism and normals as controls. Older Patients and Telethennography Studies Methods and results In an effort to estimate the number of Rett syndrome patients who were institutionalized, we visited five residential facilities. We identified six patients confOrming to
From Kennedy Institute for Handicapped Children, Baltimore (SN, MM, HM); Johns Hopkins Hospital, Baltimore (SN, SC, SU, HM) ; and UCLA, Neuropsychiatric Institute, Center for Health Science (MP). Correspondence address: Sakkubai Naidu, MBBS, Kennedy Institute, Johns Hopkins Hospital, Department of Neurology and Pediatrics, 707N. Broadway, Baltimore, MD 21205. USA.
the classical Rett syndrome phenotype. They ranged in age from 24 to 42 years. Two of them had been known to us previously. Observations in these two older Rett syndrome patients are tabulated in Table 1. Due to the recognition of early onset of cold extremities with vascular instability in this disorder thermographic studies were performed in four girls aged 5-14 year-old patients with Rett syndrome. The details of the thermographic techniques used in this study have been described elsewhere [1]. Thermography confirmed the clinical observation of distal coldness in all extremities but most severe in the lower extremities. Of particular interest are the findings in a 14-year-old girl who has an inadvertent unilateral left sympathectomy during surgery for scoliosis (Fig 2). The left leg was 3°C and the left foot was O.7°C warmer than the right. There was also a disproportion in size; the left foot was 1.5 cm longer than the right, and the width of the nail on the left big toe exceeded that on the right by2mm.
Discussion Skin temperature is a function of sympathetic vasomotor control and reflects a disturbance in peripheral nerve function. Using telethermography, direct measurement of skin temperature records segments corresponding to the known distribution of sensory and sympathetic nerves, and demarcates these areas [5]. That sympathectomy resulted in increased warmth and vascularity is not surpris-
Table 1 Neurological
Behavior
Other
1-42 yrs
Intense gaze, hyperventilation, bruxism, breath holding, hand wringing
Selective distal wasting lower legs, absent DTR's at knees, feet small and plantar flexed
Scoliosis, pneumonia, phenobarbital and dilantin, seizure free many years
11-42 yrs
Intense gaze, hyperventilation, hand wringing
Contractures and rigidity, predominant distal wasting lower legs, absent DTR's both LE.
Severe seborrhea, chronic mastitis, edentulous, no scoliosis, phenobarbital, tegretol and tranxene
ing. The increase in the size of the foot and nails compared to the opposite side 5 years later, however, raises certain important questions as to the etiopathology of the distal w'asting and cold extremities with vascular instability in this disorder_ Increased sympathetic tone needs to be considered. The preliminary observations by Percy et al [6] and Jellinger et al [7] suggest an axonopathy with increased filaments in the axons and increased clusters of small unmyelinated fibers. This axonopathy may be related to the postulated distal dysfunction of the sympathetic nervous system. There is no clear evidence
of other systemic or central sympathetic abnormalities in this disorder. The elderly Rett syndrome patients were both nonambulatory. Deep tendon reflexes were absent in the legs, but present in the upper extremities. There was severe selective wasting of the muscles of the lower leg with foot deformities. Unfortunately, detailed neurophysiologic studies could not be performed in these older patients. It has been postulated that Rett syndrome may be static after the first decade of life, Progressive increase in rigidity over a seven year period in case II and the
A
GbOse3 Car ·" :1:::',.· ~
.
~:
r;
'Yo
~ I.
~ ~,
o
, 2 34 '5 6
Fig 1 Thin layer chromatography of plasma glycosphingolipids. Suitable aliquots of freshly drawn plasma were extracted with organic solvents (2/ and glycosphingo/ipids isolated employing silicic acid column chromatography [3]. Suitable aliquots of the total glycosphingolipid fraction representing one ml of plasma were separated on HPTLC plates. Authentic standards of neutral g[ycosphingolipids and gang/iosides were co-chromato-graphed and developed simultaneously. The developing solvent was chloroform-metha'lo[-water (containing 0.02% CaCI 2 ) 60 :40: 9. Following development, the chromatogram was sprayed with aniline-diphenylamifle reagent and photographed. A. lane ], human kidney, CbOse3Cer; lane 2; CM 1 ganglioside; lane 3 and 4 CD 2 and CD10 standards; lane 5, 6 plasma glycosphingo-/ipids from two non identical twins EB. unaffected f lane 5) and KB, affected (lalle 6) . B. lane 1, human kidney CbOse3Cer; lane 2, patient (KO) clinically affected but negative, 3, 4, 5, 6 affected patient, KB, A.D., N.c., N.c., lane 7, normal human plasma.
526
Brain & De velopment, Vol 9, No 5, 1987
Fig 2 Thermogram of legs and feet of a 14-yr-old girl with Rett syndrome who had a unilateral left lumbar sympathectomy at 9 yrs of age. The temperature below the right knee was reduced; by O. 7°ein the midleg region and by 3°eat the right foot. The right foot is smaller than the left. This finding suggests that the distal wasting in Rett syndrome patients may be a reflection of increased sympathetic tone.
distal wasting in both older patients raises concerns of a slowly progressive central and peripheral nervous system disorder. The progressive loss of deep tendon reflexes in both the older cases may be a reflection of the peripheral nerve changes observed by Percy et al [6], Jellinger et al [7] and be relevant to our thermographic findings. Detailed serial neurophysiologic and neuropathological studies will help to clarify the nature of these changes. Glycolipid Studies Methods and results Ten patients between 3 1/2 to 21 years of age with the classical clinical presentation of Rett syndrome were randomly selected for the glycospingolipid (GSL) assay. Three were black, six were white, and one of East Indian origin. Four were fasting samples. Five of the ten were on various anticonvulsants for seizures. Ten cc of blood was collected in an EDT A tube and plasma was immediately obtained and kept refrigerated at 4°C until the time of assay. Random urine samples of at least 44 cc were obtained and spun to obtain cells. Total lipids were extracted from plasma organic solvents as described previously [2]. The total lipid extracts were dried in N2 atmosphere and fractionated by silicic acid column chromatography [3]. The GSL fraction were subjected to alkali-catalyzed methanolysis to remove all contaminating phospholipids and dialyzed. The total G'SL fraction were separated by high per-
formance thin layer chromatography employing chloroform-methanol-water (containing .02% CaCh) (60:40:9, V/V). One set of chromatoplates were stained with aniline-diphenylamine reagent to detect the GSLs and photographed. The second set of chromatoplates were dried and exposed to iodine vapor. Gel area co~respond ing to standard GSL and a disialo ganglioside (GD 2) were scraped, eluted with organic solvents and dried in N2 atmosphere. The HPTLC purified samples were subjected to acid-catalyzed methanolysis. Total sphingosine was extracted from the methanolysates by extraction with solvents and quantified [4]. Nine out of 10 children demonstrated a novel glycolipid that migrated in the vicinity of GD2 (disialoganglioside) but was devoid of sialic acid. This substance was present whether the patients were fasting or post prandial and whether or not anticonvulsants were being taken. The negative result occurred in a 17 -year-old girl with typical clinical features, but who was born at 32 weeks gestation and never had the normal early development so characteristic of the Rett's child. A chromatograph of the plasma samples is shown in Fig 1. Since our observations on the new glycolipid were initially promising, a blind study was then conducted with 10 Rett syndrome patients compared to equal numbers of normal girls, and girls with various types of seizure disorders on anti-convulsants. The 6 patients with autism were all boys, in whom its incidence is higher. We noted an increased association (5/10) between the abnormal glycolipid and Rett syndrome girls. One normal girl, one girl with seizures and three of the autistic boys were also positive. It should also be mentioned that difference in testing conditions did occur in the blinded study; namely the test samples analyzed in some of the younger children was small in volume and may have contributed to reduced recovery of the GSL in all groups. Also, all samples were frozen at _80°C for various lengths of time until all of the patients had been accumulated. Under these conditions the association of this glycolipid with Rett syndrome did not reach statistical significance, and therefore the specificity of this assay to Rett syndrome is not clear. Discussion While the possible association between Rett syndrome and an unusual glycolipid is a provocative finding, the data available at this time do not permit final appraisal of its diagnostic Significance or pathogenetic role. Studies are in progress to evaluate a larger group of Rett syndrome and control patients as well as to define the structure of this compound. Once this is accomplished, we aim to raise an antibody to it, and then to utilize a radioimmunoassay to evaluate its potential as a marker for Rett syndrome.
Naidu et al: Rett sy ndrome 527
ACKNOWLEDGMENTS Thi& study was supported in part by an Institutional Research Grant from Johns Hopkins University School of Medicine.
REFERENCES 1. Uematsu S, Long DM. Thermography in chronic pain. In: Uematsu S, ed. Medical thermography, theory and clinical applications. Los Angeles: Brentwood, 1976,52-68. 2. Fo1ch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lip ides from animal tissues. ] Bioi Chern, 1957;226:497-506.
528 Brain & Development, Vol 9, No 5, 1987
3. Sweeley CC, Walker B. Determination of carbohydrates in glycolipides and gangliosides by gas chromatography. Anal Chern 1964;36:1461-6. 4. Lauter CJ, Trams EG. A spectrophotometric determination of sphingosine. ] Lipid Res 1962;3: 136-8. 5. Brelsford KL, Uematsu S. Thermographic presentation of cutaneous sensory and vasomotor activity in the injured peripheral nerve. ] Neurosurg 1986;62:711-5. 6. Percy AK, Zoghbi H, Riccardi VM. Rett Syndrome: initial experience with an emerging entity. Brain Dev (Tokyo) 1985;7:300-4. 7. Jellinger K, Seitelberger F. Neuropathology of Rett syndrome. Am] Med Genet 1986;24 (suppl 1):259-88.