Sterol profiles in plasma and erythrocyte membranes in patients with Smith-Lemli-Opitz syndrome: a six-year experience.

BACKGROUND: This study reports our experience over the last six years in the diagnosis of Smith-Lemli-Opitz syndrome and other inborn errors of cholesterol biosynthesis. METHODS: Gas chromatography/mass spectrometry was used to obtain sterol profiles in plasma and erythrocyte membranes of suspected patients. RESULTS: Plasma sterol reference values calculated in unaffected subjects (n=276) were in agreement with those previously reported. Among patients investigated from 2005 to 2010, we report 16 patients affected by Smith-Lemli-Opitz syndrome, three of whom represent new cases and 13 of whom were follow-up patients. In this period we also identified a new case of chondrodysplasia punctata 2 X-linked. The estimated incidence obtained for Smith-Lemli-Opitz syndrome was 1:93 suspected patients (1.08%). We also studied the effect of storage on the dehydrocholesterols/cholesterol ratio in plasma and erythrocyte membranes of patients affected by Smith-Lemli-Opitz syndrome stored at -20°C for up to 22 and 20 months, respectively. A significant negative linear correlation between storage time and the dehydrocholesterols/cholesterol ratio was identified in both plasma and erythrocyte membranes. The decrease in the dehydrocholesterols/cholesterol ratio in erythrocyte membranes was at least two-fold higher than in plasma. CONCLUSIONS: The results of this study may be helpful for diagnosis and interpretation of data in patients with findings suggestive of a cholesterol biosynthesis defect.

Conradi-Hünermann-Happle syndrome.

A seven-year-old girl was born with red, scaly skin that later evolved into hypopigmentation and follicular atrophoderma in a widespread distribution that followed Blaschko lines. She also had patchy, scarring alopecia, left microphthalmia, bilateral cataracts, dysmorphic facies, short stature, hip dysplasia, and vertebral abnormalities. An elevated plasma 8(9)-cholestenol level confirmed the diagnosis of Conradi-Hünermann-Happle syndrome, which is caused by mutations in the emopamil binding protein (EBP) gene. This reports highlights the evolution of clinical findings over time in this X-linked dominant form of chondrodysplasia punctata.

Novel and recurrent EBP mutations in X-linked dominant chondrodysplasia punctata.

Chondrodysplasia punctata (CDP) is a heterogeneous group of skeletal dysplasias characterized by stippled epiphyses. A subtype of CDP, X-linked dominant chondrodysplasia punctata (CDPX2), known also as Conradi-Hünermann-Happle syndrome, is a rare skeletal dysplasia characterized by short stature, craniofacial defects, cataracts, ichthyosis, coarse hair, and alopecia. The cause of CDPX2 was unknown until recent identification of mutations in the gene encoding Delta(8),Delta(7) sterol isomerase emopamil-binding protein (EBP). Twelve different EBP mutations have been reported in 14 patients with CDPX2 or unclassified CDP, but with no evidence of correlation between phenotype and nature of the mutation. To characterize additional mutations and investigate possible phenotype-genotype correlation, we sequenced the entire EBP gene in 8 Japanese individuals with CDP; 5 of them presented with a CDPX2 phenotypes. We found EBP mutations in all 5 CDPX2 individuals, but none in non-CDPX2 individuals. Three of these CDPX2 individuals carried novel nonsense mutations in EBPand the other two, separate missense mutations that had been reported also in different ethnic groups. Our results, combined with previous information, suggest all EBP mutations that produce truncated proteins result in typical CDPX2, whereas the phenotypes resulted from missense mutations are not always typical for CDPX2. Patients with nonsense mutations showed abnormal sterol profiles consistent with a defect in Delta(8), Delta(7) sterol isomerase. X-inactivation patterns of the patients showed no skewing, an observation that supports the assumption that inactivation of the EBP gene occurs at random in affected individuals.

An improved method for quantification of very long chain fatty acids in plasma.

Most peroxisomal disorders can be detected via analysis of very long chain fatty acids (VLCFA) and phytanic acid in plasma or serum. Previous methods utilizing gas-liquid chromatography (GLC) alone are time consuming and are hampered by interference from cholesterol derivatives. We describe here a GLC-mass spectrometry method for the simultaneous quantification of VLCFAs and phytanic acid. The method employs single ion monitoring with deuterated internal standards. We studied 38 normal controls and 12 patients with peroxisomal diseases and found complete discrimination between the two groups. Comparison with other methodology is discussed. We believe this to be a practical and accurate method for the quantification of both VLCFAs and phytanic acid in serum or plasma. It should be useful for laboratories involved in the diagnosis of biochemical disorders.

Circulating C-type natriuretic peptide (CNP) rescues chondrodysplastic CNP knockout mice from their impaired skeletal growth and early death.

C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth through a subtype of membranous guanylyl cyclase receptor, GC-B. Although its two cognate natriuretic peptides, ANP and BNP, are cardiac hormones produced from heart, CNP is thought to act as an autocrine/paracrine regulator. To elucidate whether systemic administration of CNP would be a novel medical treatment for chondrodysplasias, for which no drug therapy has yet been developed, we investigated the effect of circulating CNP by using the CNP transgenic mice with an increased circulating CNP under the control of human serum amyloid P component promoter (SAP-Nppc-Tg mice). SAP-Nppc-Tg mice developed prominent overgrowth of bones formed through endochondral ossification. In organ culture experiments, the growth of tibial explants of SAP-Nppc-Tg mice was not changed from that of their wild-type littermates, exhibiting that the stimulatory effect on endochondral bone growth observed in SAP-Nppc-Tg mice is humoral. Then we crossed chondrodysplastic CNP-depleted mice with SAP-Nppc-Tg mice. Impaired endochondral bone growth in CNP knockout mice were considerably and significantly recovered by increased circulating CNP, followed by the improvement in not only their longitudinal growth but also their body weight. In addition, the mortality of CNP knockout mice was greatly decreased by circulating CNP. Systemic administration of CNP might have therapeutic potential against not only impaired skeletal growth but also other aspects of impaired growth including impaired body weight gain in patients suffering from chondrodysplasias and might resultantly protect them from their early death.

Highly sensitive analysis of sterol profiles in human serum by LC-ESI-MS/MS.

We have developed a highly sensitive and specific method for the analysis of serum sterol profiles. Sterols in 1 mul of dried serum were derivatized into picolinyl esters (3beta-picolinate) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using the electrospray ionization (ESI) mode. In addition to cholesterol, 19 cholesterol precursors, cholestanol, campesterol, sitosterol, and sitostanol were identified simultaneously. Quantitative analyses for the picolinyl esters of 11 available sterols were performed, and detection limits were found to be less than 1 pg on-column. Reproducibilities and recoveries of 8 noncholesterol sterols were validated according to one-way layout and polynomial equation, respectively. The variances between sample preparations and between measurements by this method were calculated to be 1.6% to 8.2% and 2.5% to 16.5%, respectively. The recovery experiments were performed using 1 mul aliquots of normal human serum spiked with 1 ng to 6 ng of sterols, and recoveries of the sterols ranged from 88.1% to 102.5% with a mean recovery of 98.1%. The present method provides reliable and reproducible results for the identification and quantification of neutral sterols, especially in small volumes of blood samples, which is useful for serological diagnosis of inherited disorders in cholesterol metabolism and for noninvasive evaluation of cholesterol biosynthesis and absorption in humans.

Conradi-Hünermann-Happle syndrome (X-linked dominant chondrodysplasia punctata) confirmed by plasma sterol and mutation analysis.

Conradi-Hünermann-Happle syndrome, or X-linked dominant chondrodysplasia punctata, is a rare genetic disorder characterized by skeletal dysplasia, stippled epiphyses, cataracts, transient ichthyosis and atrophic residua in a mosaic pattern. Mutations in the gene encoding the emopamil-binding protein have been identified as an underlying cause. A 5-year-old girl presented for evaluation of ill-defined patches of cicatricial alopecia. In addition, subtle follicular atrophoderma, esotropia, craniofacial asymmetry and short stature were noted. Her history revealed widespread scaly erythema and eye surgery for congenital cataract in the first months of life. Diagnosis of Conradi-Hünermann-Happle syndrome was confirmed by plasma sterol analysis showing markedly elevated levels of 8(9)-cholestenol and 8-dehydrocholesterol and by detection of a missense mutation (c.307G>A; p.E103K) in the emopamil-binding protein gene. We suggest that plasma sterol analysis is a reliable method of establishing the diagnosis of Conradi-Hünermann-Happle syndrome, even in patients with less striking phenotypical changes beyond infancy.

Rhizomelic chondrodysplasia punctata: 16-year follow-up of a child from birth.

A boy with rhizomelic chondrodysplasia punctata, diagnosed on the laboratory evidence of a high plasma concentration of phytanic acid and a low erythrocyte concentration of plasmalogens, has been followed from birth to the age of 16 years. The clinical pattern (somatic, skeletal and neurological) tallies with published findings in this disease. Unusual findings are the associated epilepsy, confirmed by EEG, and the long survival. CT brain scan and MRI showed cortical and subcortical atrophy but not gyral abnormalities or demyelination.

Serum lipid analysis confirms the diagnosis of X-linked dominant chondrodysplasia punctata - Conradi-Hünermann-Happle syndrome.

We present the morphological and biochemical findings in a twelve month old girl with chondrodysplasia punctata X2 - Conradi-Hünermann-Happle syndrome. This disease is characterized by limb length discrepancies, growth retardation, ichthyosis, cataracts, and punctate calcification. The diagnosis could finally be confirmed by increased concentrations of cholesterol precursors as recently found in the plasma and tissues of affected patients.

Molecular prenatal diagnosis in a case of an X-linked dominant chondrodysplasia punctata.

X-linked dominant chondrodysplasia punctata, (CDPX2-MIM302960) also known as Conradi-Hünermann-Happle syndrome, is a rare form of skeletal dysplasia that affects the skeleton, skin, hair, and eyes. The disorder is caused by mutations within the emopamil binding protein (Ebp) that functions as a delta(8), delta(7) sterol isomerase in the cholesterol biosynthesis pathway. To date, over 40 separate mutations have been reported in the Ebp gene, EBP, with no obvious correlation between the molecular defects and the severity of the clinical phenotype. We have studied a 30-year-old woman who presented in adulthood with skin, hair, and mild skeletal defects but no ocular abnormalities and have identified a heterozygous missense mutation within the third transmembrane domain of the protein. In addition, we have performed molecular prenatal testing on her unborn fetus. The results demonstrate inter-familial variability for missense mutations within the emopamil binding protein and add to the molecular data for CDPX2.

Plasma bile acids in patients with peroxisomal dysfunction syndromes: analysis by capillary gas chromatography-mass spectrometry.

Six patients with disorders of peroxisomal function have been studied. Two presented in the neonatal period with the classical features of the Zellweger syndrome, two had incomplete Zellweger phenotypes, one infantile Refsum's disease and one rhizomelic chondrodysplasia punctata. Plasma bile acid profiles were determined using capillary gas chromatography-mass spectrometry. In all patients, except the case of chondrodysplasia punctata, 27-carbon and 29-carbon bile acids were present. The compounds identified included trihydroxycoprostanic acid (THCA), dihydroxycoprostanic acid (DHCA), C24-, C25- and C26-hydroxylated derivatives of THCA, a 27-carbon acid with four nuclear hydroxy groups and 3 alpha,7 alpha,12 alpha-trihydroxy-27a,27b-dihomo-5 beta-cholestan-26, 27b-dioic acid (C29-dicarboxylic acid). THCA was present at a low concentration in the patient with infantile Refsum's disease; the concentration of DHCA and the C29 dicarboxylic acid were considerably higher. The presence of abnormal bile acids in patients with Zellweger syndrome and infantile Refsum's disease could be explained by the absence of peroxisomes from their hepatocytes. In chondrodysplasia punctata the cause of peroxisomal dysfunction must be different, since normal bile acid synthesis is preserved.

Accumulation of pristanic acid (2, 6, 10, 14 tetramethylpentadecanoic acid) in the plasma of patients with generalised peroxisomal dysfunction.

The plasma of some patients with biochemical evidence of a generalised peroxisomal dysfunction (GPD) show greatly increased levels of phytanic acid as well as its alpha-oxidation product, pristanic acid (2, 6, 10, 14-tetramethylpentadecanoic acid). Increased amounts of 14- and 16- carbon branched chain fatty acids are also found in some of these patients. As pristanic acid is present in normal or near-normal amounts in classical Refsum disease and rhizomelic chondrodysplasia, two disorders characterised by deficiencies in phytanic acid oxidation, we speculate that its accumulation is not secondary to a defect in the alpha-oxidation of phytanic acid, but is indicative of a block in the peroxisomal beta-oxidation of pristanic acid. The finding of phytanic acid, as well as a number of its metabolites in patients with inherited defects in peroxisomal biogenesis indicates that a number of the steps in phytanic acid degradation may be confined to peroxisomes.

Skewed X-chromosome inactivation causes intra-familial phenotypic variation of an EBP mutation in a family with X-linked dominant chondrodysplasia punctata.

X-linked dominant chondrodysplasia punctata (CDPX2) is a skeletal dysplasia characterized by stippled epiphyses, cataracts, alopecia and skin lesions, including ichthyosis. CDPX2 exhibits a number of perplexing clinical features, such as intra- and inter-familial variation, anticipation, incomplete penetrance and possible gonadal and somatic mosaicism. Recently, mutations in the gene encoding Delta8,Delta7 sterol isomerase/emopamil-binding protein (EBP) have been identified in CDPX2. To better understand the genetics of CDPX2, we examined the entire EBP gene by direct sequencing in four CDPX2 patients. We found EBP mutations in all four patients, including three novel mutations: IVS3+1G>A, Y165C and W82C. Surprisingly, a known mutation (R147H) was identified in a patient and her clinically unaffected mother. Expression analysis revealed the mutant allele was predominantly expressed in the patient, while both alleles were expressed in the mother. Methylation analysis revealed that the wild-type allele was predominantly inactivated in the patient, while the mutated allele was predominantly inactivated in her mother. Thus, differences in expression of the mutated allele caused by skewed X-chromosome inactivation produced the diverse phenotypes within the family. Our findings could explain some of the perplexing features of CDPX2. The possibility that an apparently normal parent is a carrier should be considered when examining seemingly sporadic cases and providing genetic counseling to CDPX2 families.

X-linked dominant chondrodysplasia punctata (CDPX2) caused by single gene mosaicism in a male.

X-linked dominant chondrodysplasia punctata (CDPX2; Happle syndrome) is recognized almost exclusively in females, who display mosaic and asymmetric features, presumed to arise secondary to random X-inactivation. CDPX2 results from mutation of an X-linked gene coding for sterol-delta(8)-delta(7) isomerase (emopamil binding protein). We describe a boy with clinical features of CDPX2 (including those presumed to arise usually secondary to functional mosaicism in females). Biochemical and molecular studies demonstrate that he is mosaic for a sterol-delta(8)-delta(7) isomerase gene mutation. He is the first reported example of single gene mosaicism giving rise to CDPX2 in a male.

Profiles of very-long-chain fatty acids in plasma, fibroblasts, and blood cells in Zellweger syndrome, X-linked adrenoleukodystrophy, and rhizomelic chondrodysplasia punctata.

Profiles of saturated very-long-chain (> C22) fatty acids were studied in plasma, fibroblasts, erythrocytes, platelets, and leukocytes of patients affected by peroxisomal disorders such as Zellweger syndrome, X-linked adrenoleukodystrophy (X-ALD), and classic rhizomelic chondrodysplasia punctata (RCDP) and in controls. In Zellweger patients, the concentration of hexacosanoic acid (C26:0) and the C26:0/C22:0 ratio are greatly increased in plasma and fibroblasts. However, the plasma concentration of docosanoic acid (C22:0) is greatly decreased. Also in platelets, leukocytes, and to a lesser extent erythrocytes, the C26:0 concentrations and both the C26:0/C22:0 and C24:0/C22:0 ratios are greatly increased. The C24:0/C22:0 ratio is significantly increased in plasma, platelets, and leukocytes, but not in erythrocytes. In X-ALD, the C26:0 concentration and the C26:0/C22:0 and C24:0/C22:0 ratios are significantly increased in plasma, fibroblasts, platelets, and leukocytes, but the erythrocytes show substantial overlap in the 5-90% ranges between controls and patients. In RCDP, slightly increased C26:0 and C26:0/C22:0 ratios are found in erythrocytes, platelets, and leukocytes, but not in plasma and fibroblasts. We conclude that plasma and fibroblasts are the specimens of choice for biochemical diagnosis of Zellweger syndrome and X-ALD, respectively. The slight increase in C26:0 in blood cells of RCDP patients suggests a decreased flux of very-long-chain fatty acids through the peroxisomal beta-oxidation pathway in liver in this genetic disorder.