In 1969, France and Liggins reported studies of a single pregnancy in which estriol production was strikingly impaired despite the normal growth and viability of the fetus.1 Labor was prolonged, and after delivery the placental extracts were assayed for activities for several enzymes thought to be important in estrogen biosynthesis. A specific and virtually complete absence of STS was found. These investigators were subsequently able to study another pregnancy in vivo by loading techniques.157 Intravenously administered DHEAS could not be converted to estrogen in a normal fashion, while exogenous free DHEA could serve as an effective substrate. This suggested a metabolic block in the conversion of DHEAS to DHEA, which was again confirmed by biochemical studies of the placenta.
During the ensuing years, a number of similar cases were described in the obstetrical literature (see reviews in references158 and 159). This was in part due to the growing popularity of the use of radioimmunoassay determination of maternal urinary and serum estriol levels as an index of fetal-placental-maternal integrity and well-being during pregnancy. In many of these earlier cases, there was an ascertainment bias, because estriol measurements were done routinely only in complicated obstetrical situations. However, with the advent of large-scale estriol screening programs, many more cases of apparent “placental” STS deficiency were identified, and, by the mid-1970s, this was a well-established clinical entity. The total 24-h urinary estrogen excretion of women carrying affected pregnancies is generally less than 3 mg, and serum estriol levels are dramatically reduced to less than 10 percent of normal values controlled for gestational age.
In the initial reports of the clinical features of STS deficiency, a relative refractoriness to the onset of labor was noted, particularly in primigravidas. Further evaluation suggests that this may be due to some difficulty in cervical effacement with prolongation of labor. Despite that many of the women carrying affected fetuses require cesarian section, many do deliver vaginally.160 Some women have had a history of prior pregnancy losses with death of full-term or postmature infants. There was one report of placental sulfatase deficiency associated with intrauterine fetal death.161 A single pregnancy in which STS deficiency was present in a fetus as the result of autosomal recessive multiple sulfatase deficiency disease also had marked impairment of estriol production.162 Diminished maternal-fetal-placental estrogen production is also seen in substrate deficiency conditions such as fetal adrenal hypoplasia (with or without anencephaly). Infants born of pregnancies complicated by STS deficiency are usually clinically normal at birth, and their placentas lack any anatomic defects. Maternal urinary excretion of a number of sulfated steroids is detectably abnormal during the latter half of pregnancy, with the 16-hydroxy-DHEAS level averaging 20 times normal.163 DHEAS levels in amniotic fluid from affected pregnancies are also strikingly increased.164,165 In contrast, cord blood levels of DHEAS and 16-hydroxy-DHEAS are usually normal in affected infants.1,158,163,166 It is hypothesized that the local estrogen/progesterone ratio in chorion and decidua influence myometrium contractility, and that this ratio is abnormal in STS deficiency.167
Most of the earlier studies of STS deficiency focused on obstetrical aspects. Because cord blood and neonatal urinary steroid levels were normal in infants born of affected pregnancies and because these children looked well clinically, it was speculated that STS deficiency was an enzymopathy that was confined to the placenta. This view changed with the development of sensitive enzyme assays, which could be applied to cultured skin fibroblasts, leukocytes, and hair follicles.168 It then became clear that STS deficiency is a generalized metabolic disturbance that affects essentially all tissues in the fetus and newborn. Furthermore, a visible phenotypic manifestation of ichthyosis made it clear that there was a specific postnatal phenotype associated with this inborn error of metabolism. The availability of the fibroblast assay has facilitated widespread case finding and the performance of family studies that would not have been possible were it necessary to have placental tissue in order to recognize affected individuals. The first reports of STS deficiency noted recurrence in families and the fact that all affected fetuses were males. Application of the cell culture assay to postnatally derived samples permitted the rigorous confirmation of X-linked inheritance in several families.169,170
STS Deficiency as the Cause of X-Linked Ichthyosis
Ichthyosis describes a number of genetic and acquired skin disorders.171 The clinical hallmark is hyperkeratosis or increased thickness of the stratum corneum (Fig. 166-7). A number of complex syndromes have been described with ichthyosis as one component. In addition, distinct hereditary disorders have been delineated in which ichthyosis is the principal feature, including the relatively severe autosomal recessively inherited conditions known as lamellar ichthyosis and congenital ichthyosiform erythroderma, the somewhat milder disorder autosomal dominant ichthyosis vulgaris, various types of autosomal dominant bullous ichthyosis, and X-linked ichthyosis. Table 166-2 lists the features that should be considered in the differential diagnosis of the major types of ichthyosis. Reviews on the clinical, histopathologic, and biochemical features of the ichthyoses are available.172–175
Photograph of the arm of a 45-year-old adult with X-linked ichthyosis due to STS deficiency. The scales are thick and dark and are present on the neck, trunk, and lower soles. This individual had two affected grandsons and an affected brother and uncle.
Table 166-2: Differential Diagnosis of the Major Types of Ichthyosis |Favorite Table|Download (.pdf) Table 166-2: Differential Diagnosis of the Major Types of Ichthyosis
|Disorder ||Inheritance* ||Incidence ||Onset/Natural Course ||Scale ||Distribution ||Associated Features ||Histology ||Biochemical Findings |
|Ichthyosis vulgaris ||AD ||1:250 ||3–12 months, improves with age ||Fine, like bran flakes, mostly adherent ||Extensor extremities, palms and soles: face spared ||Keratosis pilaris, atopy ||Thickened stratum corneum; diminished or absent granular layer ||Unknown |
|X-linked ichthyosis ||XL ||1:2000–6000 (males) ||0–3 months, improves with age ||Large, dark, adherent ||Both extensor and flexor extremities, trunk, lateral face, neck; palms and soles spared (in most cases) ||Corneal opacities, cryplorchidism, testicular cancer ||Thickened stratum corneum; normal to thickened granular layer ||Steroid sulfate deficiency; abnormal, mobility of serum β lipoprotein increased cholesterol sulfate in serum and stratum corneum |
|Lamellar ichthyosis ||AR, AD (rare) ||1:300.000 ||birth, persistent ||Large, thick, plate-like, raised borders ||Generalized, palms and soles ||Ectropion, eversion of lip; collodion baby; hyperpyrexia ||Hyperkeratosis; granular layer present ||Increased free sterols and ceramides stratum corneum |
|Congenital ichthyosiform erythroderma ||AR ||1:300.000 birth, persistent ||Fine white scale ||Generalized ||Erythroderma, collodion baby; scarring alopecia; nail dystrophy ||hyperkeratosis; granular layer present ||Unknown |
|Bullous ichthyoses ||AD ||1:300.000 ||birth, may improve with age ||Verrucous, thick, dark ||Generalized with blisters, accentuated in flexures, face spared ||Generalized bacterial overgrowth; foul body odor; nail dystrophy ||Vacuolization of the granular and upper epidermal cell layers with variable keratosis |
It has been known for more than 100 years that ichthyosis can segregate in some families as an X-linked trait.164 The work of Wells, Kerr, and Jennings in the early and middle 1960s provided clear genetic and clinical delineation of these various forms of ichthyosis.165,176– 179 These workers were able to demonstrate that autosomal dominant and X-linked ichthyosis were relatively common disorders, with the latter condition being present in approximately 1 in 6000 males studied in their population. They had reason to believe that they had complete ascertainment within the geographic area under consideration. They were able to show that X-linked ichthyosis differed from autosomal dominant ichthyosis vulgaris in age of onset, relative distribution of disease, severity, and a variety of histopathologic features.
The clinical features of X-linked ichthyosis are listed in Table 166-3. Aside from the obvious differences in inheritance patterns, X-linked ichthyosis is characterized by onset between birth and 4 months of age and involvement of the upper and lower limbs and trunk. There is frequent involvement of the scalp and neck but, in most cases, the palms and soles are spared. The nails and hair are normal. The scales are large, dark, and prominent. Histologically, there is hyperkeratosis, with a normal or increased granular layer. Finally, characteristic corneal opacities may be observed on slit-lamp examination. These opacities are found in Descemet's membrane or in the deep stroma anterior to it (Fig. 166-8), and they have no effect on visual acuity.174,180– 182 A survey of 38 patients with X-linked ichthyosis and of female heterozygotes found an incidence of corneal opacities of approximately 25 percent in both groups.181 Aside from the eye findings, most female heterozygotes are asymptomatic and do not show any cutaneous abnormalities.
Table 166-3: Clinical Abnormalities Associated with STS Deficiency |Favorite Table|Download (.pdf) Table 166-3: Clinical Abnormalities Associated with STS Deficiency
|Prenatal ||Postnatal |
|Consistent Features || |
|Low maternal urinary and serum estriol ||Ichthyosis with onset from birth to 3–4 months |
|Elevated DHEAS and 16-hydroxy-DHEAS in maternal urinary and amniotic fluid ||Corneal opacities |
|Absent placental STS activity ||Increased cholesterol sulfate in plasma, red blood cells, and skin with rapidly migrating LDL |
| ||Absent STS activity |
| Variable Features || |
|Delayed onset of labor ||Undescended testis |
|Relative refractoriness of cervical dilatation ||Testicular tumors (?) |
Eye lesions in X-linked ichthyosis. Corneal opacities associated with X-linked ichthyosis are found in Descemet's membrane or in the deep stroma anterior to Descemet's membrane. (From Ghadially and Chong. 174 Used by permission of Dermatologic Clinics.)
Two groups independently observed that patients with STS deficiency, as ascertained by low estrogen production, had clinically apparent ichthyosis.169,170 Furthermore, in a number of the extended families of probands, a clear history of X-linked ichthyosis could be obtained. When family studies of STS activity in cultured fibroblasts were conducted, complete concordance between enzyme deficiency and ichthyosis was demonstrated. Finally, many studies of families ascertained solely on the basis of the X-linked inheritance of ichthyosis showed them all to be associated with STS deficiency, while enzyme assays done on patients with other types of ichthyosis revealed normal activity levels.183,184
It was suggested that STS deficiency might not be causally related to ichthyosis but that the STS gene and the “ichthyosis” gene might be contiguous and included in deletion events. Although STS deficiency is often the result of a gene deletion (see “The Molecular Basis of STS Deficiency” below), it is clear that STS deficiency per se does cause the ichthyosis. There are several reasons for coming to this conclusion. First, there is an invariant association between ichthyosis and STS deficiency. All patients lacking STS activity have this phenotypic finding. Second, there is good pathophysiological reason to suspect that disordered sulfated sterol metabolism might interfere with epidermal function (see “Ichthyosis and Lipid Metabolism” below). The third line of evidence supporting the causal relationship of X-linked ichthyosis and STS deficiency is genetic. At least seven patients with point mutations in the STS gene,185– 188 and an additional subject with an entirely intragenic deletion at the STS locus,58 have ichthyosis. In addition, patients with the rare autosomal recessive disorder multiple sulfatase deficiency have reduced STS activity as the result of a non-X–linked mutation, and still have clinically apparent ichthyosis. Finally, somatic transfer of the STS gene to keratinocytes from patients with STS deficiency leads to a partial correction of the phenotypic abnormalities observed in tissue culture and in xenotransplants.189,190 An exceptional pedigree with XLI was published by Robledo et al.191 Affected individuals from this pedigree show the classical clinical features of XLI but appear to have normal levels of STS enzymatic activity. Consistently, molecular analysis of patients' DNA failed to detect any abnormality of the STS gene. Partial linkage data appear to exclude linkage of the disease locus to markers from the Xp22.3 region, suggesting that XLI may also be caused by mutation of another gene located in a different region of the X chromosome.191
To date, the only sulfated 3β-hydroxy steroid observed to accumulate in STS-deficient patients in amounts comparable to the increases of substrates seen in other inborn errors of metabolism is CS. CS is strikingly elevated in plasma, red blood cell membranes, and the stratum corneum of STS-deficient individuals.192,193 The amounts may be as much as 20 times the normal levels. Determination of plasma CS content by either radioimmunoassay,192,193 gas chromatography,194 or HPLC/mass spectrometry195 may be of use for diagnostic purposes. As in normal individuals, most of the CS is physically associated with the LDL plasma fraction and gives the LDL an abnormal electronegativity. In patients with STS deficiency, an increase of the CS content leads to an abnormal electrophoretic mobility of LDL.193 A systematic study of LDL mobility in 14 patients with STS deficiency and in normal individuals, demonstrated that LDL migrated consistently faster in patients with STS deficiency than in normal individuals.196 The relative contents of apoB, cholesterol, and triglyceride were also altered in LDL from patients with STS deficiency. Female heterozygotes and individuals with autosomal dominant ichthyosis vulgaris showed no abnormalities. This and other studies194 suggest that lipoprotein electrophoresis could also represent a simple, readily available screening test for X-linked ichthyosis.
Ichthyosis and Lipid Metabolism
The end product of epidermal differentiation is the stratum corneum, which functions primarily as a barrier against transepidermal water loss.197 This barrier function is provided by a unique two-compartment system composed of protein-enriched corneocytes that are “glued” together by a hydrophobic, lipid-enriched intercellular matrix. Corneocytes, desmosomes, hydrolytic enzymes, and both locally synthesized and essential lipids are critical components of the stratum corneum.197 Disorders affecting any of these components may cause inflammation, hyperkeratosis, and scaling.198,199
Under normal conditions, epidermal desquamation is a continuous process resulting from detachment of individual corneocytes from the skin surface. It is known that alterations of the stratum corneum lipid content cause abnormal desquamation and scaling. In this pathologic condition, corneocytes are shed in clusters that appear as scales (the term ichthyosis derives from the Greek ichthys, “fish”).197– 199
Several studies demonstrated that drug-induced imbalances of lipid metabolism may cause scaling. Topical application of cholesterol sulfate to hairless mice can induce ichthyosis.200 Drugs inhibiting de novo cholesterol synthesis, such as lovastatin, triparanol, and nicotinic acid, have been shown to produce scaling and abnormalities of barrier function in experimental animals.197,199,201 However, the precise pathogenetic sequence of this phenomenon has not been determined. For the most part, the cholesterol, sphingolipids, and most of the fatty acids in the stratum corneum are synthesized locally by the epidermis. However, essential fatty acids, such as linoleic acids derive from dietary sources and reach the skin through systemic delivery. Elimination of these essential lipid components from the diet can cause scaling. Experimental fatty acid deficiency caused by elimination of linoleic acid from the diet causes abnormal barrier function, scaling, and alopecia. These are the consequences of alterations in the fatty composition of stratum corneum lipids, such as the substitution of oleic acid for linoleic acid in epidermal acylsphingolipids (see reviews in references197 and 199).
X-linked ichthyosis caused by STS deficiency is an example of a human-inherited lipid abnormality resulting in a scaling disorder. Studies of the lipid content of skin scales from individuals with STS deficiency revealed a fivefold increase in cholesterol sulfate and a 50 percent decrease in free sterol content.13 It has been shown that cholesterol sulfate inhibits cholesterol synthesis.16 STS may, therefore, act as a regulator for epidermal sterologenesis. It is not clear whether scaling in STS deficiency is caused by an alteration of the ratio of cholesterol sulfate to cholesterol or by an overall reduction of free sterol content. Studies of the [3H]thymidine labeling index in the epidermis of patients with STS deficiency gave normal results, suggesting that scaling is not due to hyperproliferation but to delayed desquamation.202 However, in vitro studies using fluorescence-activated cell sorting combined with [3H]thymidine labeling revealed a significant increase in the labeling of S-phase undifferentiated cells in cultures from patients with either X-linked or autosomal dominant ichthyosis, indicating the presence of hyperproliferation.203 This was particularly evident in cultures in which regeneration was induced by stripping of the suprabasal cells from the multilayered culture. These in vitro data are in conflict with the in vivo data and might result from the lack of hyperkeratosis under in vitro conditions. Nevertheless, the in vitro system is an excellent model for studying keratinocyte differentiation.203 Recent studies indicate that restoration of STS activity in cells from patients with STS deficiency after transfection with a recombinant STS gene causes an increase in the number of differentiated cells and, therefore, a shift toward a normal epidermal maturation pattern.189 Freiberg et al. developed a model of corrective STS gene delivery in vivo. Primary keratinocytes from STS-deficient patients were transduced using a new retroviral expression vector containing the STS cDNA. Transduced XLI keratinocytes were then grafted onto immunodeficient mice and regeneration of a full thickness epidermis, histologically indistinguishable from that formed by normal keratinocytes, was achieved. Transduced XLI epidermis also showed a return of barrier function parameters to normal.190 These experiments represent the first attempt to genetically correct an inherited defect of keratinocytes.
Ichthyosis is also found in other genetic disorders of lipid metabolism (Table 166-4).204 Refsum syndrome, in which ichthyosis is a prominent feature, is the result of a defect in phytanic acid oxidation with the accumulation of phytanic acid cholesterol esters.204 Abnormal fatty alcohol metabolism has been reported in Sjögren-Larsson syndrome, in which ichthyosis also occurs,205 and harlequin ichthyosis fetuses have been found to have various abnormalities of epidermal lipids.206
Table 166-4: Genetic Disorders of Lipid Metabolism Producing Ichthyosis |Favorite Table|Download (.pdf) Table 166-4: Genetic Disorders of Lipid Metabolism Producing Ichthyosis
|Disorder (Chap.) ||Enzyme Defect ||Pathogenesis ||Inheritance ||Other Features |
|X-linked ichthyosis (166) ||Steroid sulfatase deficiency ||Elevated cholesterol sulfate in stratum corneum ||X-linked recessive ||See text |
|Refsum syndrome (132) ||Phytanic acid oxidase deficiency ||Accumulation of phytanic acid and Phytanic acid–cholesteryl esters ||Autosomal recessive ||Retinitis Pigmentosa, polyneuritis, ataxia, deafness |
|Sjōgren-Larsson syndrome (98) ||Fatty alcohol oxidoreductase deficiency ||Accumulation of long-chain fatty alcohols ||Autosomal recessive ||Spasticity, mental retardation, short stature, brittle hair, hypoplasia of teeth, metaphyseal dysplasia |
|Neutral lipid storage disease* ||Unknown ||Triglyceride storage of unknown cause ||Autosomal recessive ||Cataracts, deafness, ataxia, droplets In many cells |
|Multiple sulfatase deficiency (149)* ||Conversion of Cys to 2-amino-3oxopropionic acid ||STS deficiency (along with other sulfatase deficiencies) ||Autosomal recessive ||Neurodegeneration, hepatosplenomegaly, and skeletal disorders |
|Harlequin ichthyosis ||Unknown ||Increased cholesterol and triglycerides in stratum corneum ||Uncertain ||Ectropion, malformations, absent eyebrows and lashes, ? developmental delay |
STS Deficiency in Patients with Contiguous Gene Syndromes
Following the demonstration that STS deficiency is a systemic disorder affecting all tissues and organs, several centers undertook a systematic search for phenotypic and biochemical abnormalities. In addition to the ichthyosis and corneal opacities already mentioned, several patients with STS deficiency have a complex phenotype characterized by the association of multiple monogenic X-linked disorders. These complex phenotypes are the hallmark of a contiguous gene syndrome207 resulting from deletions or translocations involving the Xp22.3 region and spanning the STS gene (see Chap. 65).90,208 Some of these chromosomal rearrangements are detectable by cytogenetic analysis, although an appreciable percentage of them are submicroscopic and can only be detected by molecular analysis.90 Table 166-5 shows the full spectrum of clinical features of Xp22.3 contiguous gene syndromes. The single-gene phenotypes include short stature steroid sulfatase (SS), X-linked recessive chondrodysplasia punctata (CDPX), mental retardation (MRX), X-linked ichthyosis (XLI), Kallmann syndrome (KAL), and X-linked ocular albinism (OA1) (see review in reference90 and Chap. 65).
Table 166-5: Spectrum of Clinical Manifestations in Patients with Xp22.3 Contiguous Gene Syndromes Associated with STS Deficiency |Favorite Table|Download (.pdf) Table 166-5: Spectrum of Clinical Manifestations in Patients with Xp22.3 Contiguous Gene Syndromes Associated with STS Deficiency
|Disease Entities ||Main Features |
|Short stature (MIM312865) ||Short stature |
|X-linked recessive chondrodysplasia punctata (MIM302950) ||Nasal hypoplasia, focal calcification of cartilage, distal phalangeal hypoplasia |
|X-Linked mental retardation (MIM309530) ||Mental retardation, usually mild to moderate |
|X-Linked ichthyosis (MIM308100) ||Scaly skin |
|X-Linked Kallmann syndrome (MIM308700) ||Hypogonadotropic hypogonadism and anosmia |
|X-linked ocular albinism (MIM300500) ||Impaired visual acuity, nystagmus, strabismus, photophobia |
Figure 166-9 shows the phenotype of a patient with X-linked ichthyosis and Kallmann syndrome, carrying a deletion of both genes. The recognition of a contiguous gene syndrome has important clinical implications. A patient with STS deficiency showing a complex phenotype should be carefully examined to rule out the presence of clinical manifestations of all the other diseases listed in Table 166-5. In addition, cytogenetic and molecular analysis will determine which of the Xp22.3 disease genes is involved in the deletion event. In some cases, this has helped predict the final outcome of the phenotype in young patients.208
A boy affected by X-linked ichthyosis associated with Kallmann syndrome. The presence of dark brown scales on the skin is a feature of X-linked ichthyosis due to STS deficiency. Micropenis and microtestes are features of Kallmann syndrome and are the result of hypogonadotropic hypogonadism due to gonadotropin releasing hormone deficiency. This patient was completely unable to smell.237
In addition to the clinical implications, the study of individuals with Xp22.3 contiguous gene syndromes has been extremely valuable for the mapping of several disease loci and for the positional cloning of the Kallmann syndrome (Chap. 225), chondrodysplasia punctata and ocular albinism genes.46,135,136,209 The consensus order of disease loci in this region is this: telomere-SS-CDPX-MRX-STS-KAL-OA1-centromere.
STS Deficiency and Androgen Metabolism
In addition to the above-mentioned association of STS deficiency with Kallmann syndrome, which is characterized by hypogonadotropic hypogonadism and inability to smell (anosmia), several gonadal abnormalities have been identified in some patients who appeared to have the common type of STS deficiency and X-linked ichthyosis. At least four such patients with testicular neoplasms have been seen, including one unfortunate man with an embryonal cell carcinoma and seminoma discovered five years apart.210,211 What the precise relationship between these tumors and STS deficiency might be is unknown. In addition, Lykkesfeldt et al. observed testicular cancer in three patients with ichthyosis and normal STS activity.211
Traupe and Happle have called attention to the possible association of cryptorchidism and STS deficiency.212 They observed unilateral or bilateral testicular maldescent in 7 of 25 patients examined and found a total of 30 cases of cryptorchidism with STS deficiency described in the literature. One other series of patients has also shown this association.210 It may be hypothesized that abnormal STS function per se is responsible in some cases for hypogonadism and cryptorchidism. The relative importance of STS in testicular testosterone biosynthesis has not been definitively established; it could conceivably be implicated. It is possible that there is some degree of polymorphism in the flux through various potential steroidogenic pathways, and while testosterone biosynthesis appears to be normal in most STS-deficient patients studied, there may be a subset of patients in whom it is not, with consequent testicular abnormality. Alternatively, at different stages of ontogeny, the relative importance of various steroidogenic pathways may change.
There is probably no gross alteration of testosterone biosynthesis or secretion in STS deficiency.41,42 Several groups have measured serum levels of testosterone, follicle stimulating hormone, and luteinizing hormone in adults with STS deficiency and have found these parameters to be normal. Furthermore, several subjects who received human chorionic gonadotropin (hCG) stimulation responded with abnormal augmentation of testosterone levels. DHEAS, 17-hydroxy-pregnenolone sulfate, and androstenediol sulfate are moderately increased in the circulation of STS-deficient individuals, and their corresponding free steroids are, on average, slightly reduced.43 This suggests that STS has some physiological role in the desulfation of these steroids, but, quantitatively, the production of the free steroids must not be terribly dependent on sulfated intermediates. Conversely, there must be active systemic pathways capable of disposing of or metabolizing the sulfated compounds without the necessity for hydrolysis.
Although the quantitative importance of desulfation in the metabolism of DHEAS cannot be great, the question can be raised as to whether any desulfation of this major adrenal secretory product can be demonstrated in vivo. To clarify this issue, [3H]DHEAS and [14C]DHEA have been administered to a series of STS-deficient subjects and controls.43 The conversion of [14C]DHEA to either DHEAS or DHEA glucuronide occurred normally in STS-deficient patients, indicating no impairment of conjugation. Furthermore, the plasma half-life of [3H]DHEAS infused into STS-deficient patients was essentially normal. However, when the conversion of DHEAS to DHEA glucuronide was examined, an unexpected result was obtained. A significant amount of labeled DHEAS was converted to the glucuronide in two of three STS-deficient individuals. Presumably, the exchange of sulfate for glucuronide at the 3β-hydroxy position requires desulfation to the free intermediate and reconjugation, although it is possible that a “transconjugase” activity might exist, which could carry out this exchange without hydrolysis.
Milone et al. used gas chromatography to measure the serum levels of CS and DHEAS in normal individuals and in 15 patients with STS deficiency.194 A significant increase of both CS and DHEAS was observed in all the individuals with STS deficiency (p<0.001). They pointed out that inconsistencies of DHEAS levels described in previous studies could be due to the much greater spread of the values obtained by radioimmunoassay compared to gas chromatography, the latter being a much more reliable method for measuring DHEAS.194 The observation that some STS-deficient subjects can apparently hydrolyze some DHEAS in vivo can be explained by the presence of some cryptic STS activity situated in an anatomic site not previously anticipated. Because most patients with STS deficiency have gene deletions, it is unlikely that there are tissue differences in the expression of the authentic STS gene product. However, it is possible that there are separately encoded enzymes capable of hydrolyzing DHEAS that are expressed in a tissue-specific fashion. There is no evidence to support a second STS gene or enzyme, but a detailed autopsy survey of STS activity in organs and tissues of STS-deficient subjects has not been undertaken.
The Molecular Basis of STS Deficiency
The large majority of patients with STS deficiency do not have microscopically detectable cytogenetic abnormalities. Typically, these patients are affected by the “classical” type of STS deficiency, which is characterized exclusively by the X-linked ichthyosis phenotype. The cloning of human STS cDNAs and the characterization of the STS gene provide the basis for elucidating the molecular defects in these individuals.
Hundreds of independently ascertained patients with classical STS deficiency have been tested by Southern blotting analysis using both partial and full-length STS cDNA probes and various DNA markers flanking the gene.56,58,78– 80 213– 215 The large majority (85 to 90 percent) of these subjects, regardless of their ethnic origin, have submicroscopic deletions detectable by either Southern blotting or polymerase chain reaction (PCR) analysis.58,214 Figure 166-10 shows the results of Southern blot analysis and of a multiplex PCR test for the detection of STS gene deletions.216 Most of these deletions span the entire 146-kb STS gene and flanking sequences on Xp22.3 and are also detectable by flow cytometry.217 Two patients have been identified with partial deletions of the gene.58,214 In the first case, the deletion starts within intron 7 of the gene and extends over 150 kb downstream toward the centromere, spanning the last three exons of the gene (Fig. 166-10).214 The other patient has an intragenic deletion of about 40 kb including exons II-V. This subject's cells made reduced amounts of shortened RNA transcripts.58
DNA analysis on patients with STS deficiency. Panel A: Southern analysis of EcoRI-digested DNA samples from 11 patients (lanes 1 to 11) and one male control (lane 12) using an STS full-length cDNA clone. Panel B: Multiplex DNA amplification of the STS gene (STS-PCR) in 11 patients with STS deficiency (lanes 1 to 11, loaded in the same order as in A) and one male control (lane 12). Band a corresponds to an exon of the DMD gene and was used as an internal control for the amplification. Bands b and c correspond to the 3′ and the 5′ ends of the STS gene, respectively. MW = molecular weight marker. Panel C: Sequence of oligonucleotide primers for PCR amplifications of STS. F = forward primer; R = reverse primer. (From Ballabio et al.216 Used by permission of Human Genetics.)
A minority of patients with STS deficiency (10 to 15 percent) do not show any abnormality with Southern blotting analysis using the full-length STS cDNA probe. In seven of these patients, point mutations in the coding region of the STS gene were identified. These were a nonsense mutation at nucleotide 1236; a change of a serine to a leucine at codon 1243 (S1243L); a change of a tryptophan to an arginine at codon 1335 (W1335R), which changes a hydrophobic to a basic hydrophilic amino acid; a change of a tryptophan to a proline at codon 1336 (W1336P); a splice site mutation at nucleotide 1477; a change of a histidine to an arginine at codon 1552 (H1552R); and a change of a cysteine to a tyrosine at codon 1558 (C1558Y), which potentially prevents a disulfide bond.185,187,188 These mutations may allow insights into functionally important domains of the protein. All of these mutations resulted in lack of a functionally active STS polypeptide when expressed in COS7 cells. The mutations also appeared to affect mRNA stability.
All patients with STS deficiency tested so far, including both deletion and “nondeletion” cases, display absence of cross-reacting material by Western blot analysis using anti-STS polyclonal antibodies on patients' fibroblast extracts.72,214,218 Finally, one patient with alleged partial deficiency of STS activity has been reported.53
With the possible exception of the α-thalassemias (Chap. 181) and del(22)(q11.2q11.2) causing DiGeorge/velocardiofacial syndrome (Chap. 65), gene deletions in STS deficiency represent the highest frequency of deletion among mutations reported at a single Mendelian disease locus. In the case of the α-thalassemias, it is likely that the original mutations occurred relatively rarely but were expanded in frequency in certain populations by selection. STS deficiency is a common and equally frequent disorder in many racial and ethnic groups and geographic areas. Furthermore, no obvious selective advantage for homozygosity or heterozygosity has been suggested. Thus, it is tempting to speculate that the lesions involving the STS locus are fairly frequent de novo events. Most patients with STS deficiency carry deletions of approximately 2 Mb of DNA. It is remarkable that nullisomy of such large regions of the human genome in male individuals is viable and results in such a relatively mild clinical condition as X-linked ichthyosis. In addition to the STS gene, these deletions involve at least one other gene, GS1 , whose function is still unknown.137
Two families of low-copy-number repeats, the G1.3 and the CRI-S232 families, have been found to be interspersed in the Xp22.3 region and clustered on either side of the STS gene.219– 221 This suggested that the frequent occurrence of interstitial deletions in this region was due, at least in some patients, to abnormal pairing and homologous recombination between different repeat units.220,222 This hypothesis was strongly supported by the finding of CRI-S232 repeat units at the deletion breakpoints of most patients with STS deficiency.222 It is difficult to establish whether these unequal recombination events occur during meiotic or mitotic cell divisions of primordial germ cells, or whether they involve recombination between two discrete chromosomes, between sister chromatids, or between CRI-S232-like sequences on the same chromatid. Sequence analysis of several of these repeat units revealed the presence of two elements containing a variable number of tandem repeats (VNTR) in each of the units.223 CRI-S232 repeats are also present on the long arm of the human Y chromosome (Yq11.2). The main difference between the X-linked and the Y-linked CRI-S232 repeats is the great variation in the number of elements of the X-linked units versus the constant nature of the Y-linked ones. Furthermore, the Y-linked units appeared to have a much higher intrarepeat sequence variation.
Although most STS gene deletions appear to be due to unequal recombination between CRI-S232 repeats, there is evidence that this mechanism does not apply to all STS deletions. Physical mapping studies indicate that at least in some cases the deletion breakpoints are not located near CRI-S232 repeat units. In two of these cases the deletion breakpoint was cloned and sequenced. A 3-bp homology was found at the site of the deletion junction in one case,224 while no repeat sequences were identified in the second case (P. Yen and A. Ballabio, unpublished data).
Some patients with STS deficiency have detectable cytogenetic abnormalities, which include large terminal deletions and X/Y translocations resulting in loss of the terminal Xp region. As mentioned previously, Xp22.3 rearrangements may result in contiguous gene syndromes in male individuals. Female individuals carrying these rearrangements have terminal Xp monosomy and typically do not have any of the recessive diseases described in affected males because they have a normal X chromosome. However, almost invariably they have short stature. This observation is consistent with the putative presence of a gene affecting height in the pseudoautosomal region, two active copies of which are needed for normal growth.208,225,226 It has been proposed that this gene is the homeobox-containing gene, which maps to this region.133,134
Many patients with X/Y translocations involving breaks in Xp22.3 have been described (see reviews in references90 and 227). These translocations can be divided into two major groups: Xp/Yq and Xp/Yp translocations. Xp/Yq translocations are the most common type of X/Y translocation, and some multigenerational families segregating this chromosomal abnormality have been reported. These translocations can be recognized cytogenetically because of the presence of the highly fluorescent Y heterochromatic region (Yq11.2-qter) located at the distal Xp region. Male subjects carrying these rearrangements have no normal X chromosome, but do have an additional intact Y chromosome.
In some Xp/Yq translocations, abnormal pairing and exchange within a 1 to 1.5 Mb region of close sequence identity shared by Xp22.3 and Yq11.2 takes place. The result is a translocation of Yq11.2-qter to distal Xp with the concomitant production of a terminal Xp22.3-pter deletion. Sequence analysis of the X/Y junction fragment from two of these translocations suggests the occurrence of abnormal pairing and homologous recombination between Xp and Yq homologous regions.228,229 This distinctive mechanism may play an important role in most Xp/Yq translocations, although other mechanisms, such as nonhomologous recombination, may also be involved.
Xp/Yp translocations usually involve the testis-determining factor (TDF) on Yp and are found in sex-reversed individuals (XX males). These are probably the result of unequal exchanges within and outside the pairing region of the human sex chromosomes.230 Usually these translocations are not visible by cytogenetic analysis and can therefore only be detected using molecular probes. The breakpoint on the X chromosome in most individuals carrying this type of X/Y translocation is distal to the STS gene, leaving two intact copies of the STS gene, one on the normal and one on the derivative X chromosome. However, in rare cases, the X breakpoint may lie proximal to STS with a consequent loss of the STS gene from the derivative X chromosome.231– 233
Terminal deletions involving the STS gene can be detected by both cytogenetic and molecular analysis (see review in reference90). The name terminal deletion may not be used in a strict sense in these cases, because the deleted Xp arm presumably retains a functional telomere, which may or may not correspond to the normal Xp telomere. However, chromosomes in which Xp subtelomeric markers are deleted are usually referred to as “terminal deletions.” The position of the breakpoints in the various patients with terminal deletions, and consequently the sizes of the deleted regions, are highly variable. The molecular mechanisms underlying these types of Xp rearrangements are unknown.
The study of patients with chromosomal rearrangements in the Xp22.3 region has been essential for the construction of deletion maps of the region.91 This has made possible the ordering of several DNA markers and of the six disease loci mentioned previously. It could be anticipated that other genes in the region will be isolated as the map becomes more refined. The large number of patients with complex phenotypes due to various types of chromosomal rearrangements makes the distal short arm of the human X chromosome a very attractive region for positional cloning of disease genes. A close interaction between the clinician, the cytogeneticist, and the molecular biologist is essential in these efforts. The successful cloning of disease genes will then be of great help for the understanding of the molecular mechanisms involved in the disease pathogenesis and would possibly provide new diagnostic and therapeutic tools. Figure 166-11 shows a PCR-based diagnostic test for Xp22.3 contiguous gene syndromes.
PCR test for Xp22.3 CGS. The order of some of the disease genes and DNA markers in the Xp22.3 region is shown at the top. PAR = pseudoautosomal region; PABX = pseudoautosomal boundary on the X chromosome. A seven loci-PCR test has been designed for the detection and partial characterization of Xp22.3 CGS. a, Pattern observed in most males with isolated X-linked ichthyosis; b, pattern observed in most males with X-linked ichthyosis associated with Kallmann syndrome; c, pattern observed in males with very complex phenotypes, including features of all Xp22.3 diseases; and d, normal male pattern.
Incidence, Clinical Diagnosis, Carrier Identification, and Prenatal Diagnosis
STS deficiency a common inborn error of metabolism. It has been described in all racial backgrounds and in a diversity of ethnic populations. Frequency estimates reported by Wells and Kerr, who identified patients on the basis of X-linked ichthyosis, were about 1 in 6000 males.176 There may have been some biases in these families and others because of the use of ichthyosis as the discriminant, which could lead to underreporting. First, in cases where no extensive pedigree information is available to support a diagnosis of an X-linked disorder, dermatologists who are consulted might be less inclined to consider this diagnostic possibility. Second, because of the non-life–threatening nature of the condition, its variation in severity, seasonal fluctuations, and the lack of responsiveness (until recently) to therapy, many affected individuals have probably not sought medical care. Lykkesfeldt et al. have suggested that when patients are identified through maternal estriol screening, the incidence may be as high as 1 in 2000 males in Western Europe.234 This seems to apply also to the U.S. population, because an increased ascertainment has been observed as a result of the antenatal triple marker (including estriol) screening test. The true population incidence needs to be determined through suitable large-scale prospective studies. Similarly, the relative homogeneity or heterogeneity of mutations at this locus requires prospective assessment by molecular studies to determine whether mutations have arisen on a common haplotype background and whether deletion end points are unique or recurrent. No studies have been performed on the origin of new mutations in families with STS deficiency because sporadic cases of STS deficiency are extremely rare.
Confirmation of a suspected diagnosis is now relatively straightforward. Affected males can have STS activity assayed using one of a variety of substrates in fibroblasts, leukocytes, hair follicles, etc. Because 85 to 90 percent of patients have submicroscopic gene deletions, DNA testing is a rapid and effective way to establish a diagnosis in many instances, although STS deficiency obviously cannot be excluded on the basis of a normal result. Simple PCR tests are available for isolated STS deficiency (Fig. 166-10) and for patients with contiguous gene syndromes involving STS (Fig. 166-11).216 Molecular detection of STS gene deletions can also be performed by fluorescence in situ hybridization (FISH) on metaphase spreads using genomic clones (usually cosmids) from within the STS gene as probes.235 This method is particularly useful for the detection of female heterozygotes. A frequent XmnI RFLP has also been identified in the STS gene and can be used for family studies.236
Assessment of substrate levels may also be of use because elevated plasma CS levels do appear to be diagnostic for STS deficiency.192– 195 The finding of abnormal lipoprotein electrophoresis caused by incorporation of CS into LDL is also of great value because of the wide availability of this test in clinical laboratories.193,196 Heterozygote identification is somewhat more problematic. While it is clear that there is normally a gene dosage effect on STS enzyme activity between subjects bearing one X-encoded copy of STS (XY, XO, or XX heterozygotes for STS mutations) and those with two copies (XX), there is considerable overlap between these two groups. This is largely the result of poorly understood variations in enzyme activity measurements. Thus, it may be difficult to be absolutely certain about heterozygote status in any single individual. FISH is clearly the best method for carrier identification if a deletion has been identified in the proband.235
As with postnatal diagnosis, many methods are potentially applicable to the prenatal diagnosis of STS deficiency. Syncytiotrophoblast tissue is normally very rich in STS activity, and this is particularly true in the first trimester, when STS specific activity is highest. Thus, chorionic villus biopsy can be performed for early prenatal assessment. Again, when a deletion has been identified in the proband, PCR testing and/or FISH can be performed. Mid-trimester diagnosis is also readily made by careful assessment of serum estriol levels (using gestational-age–appropriate standards) and by measurement of amniotic fluid DHEAS concentrations. Cultured amniocytes normally express STS and can be used for either DNA or enzymatic analysis. Finally, in the third trimester, maternal urinary estriol and sulfated steroid excretion can be monitored in addition to the other studies already described. The purpose of establishing a third-trimester diagnosis of STS deficiency might be to exclude a more serious cause of incidentally detected low estriol levels. In addition, it might be argued that such diagnostic information would be of use in obstetrical management, so that prolonged labor might be anticipated and suitable intervention planned if needed.
Once a diagnosis of STS deficiency is made, a critical issue, particularly in prenatal and early postnatal diagnosis, is to rule out the presence of a contiguous gene syndrome, because this has very important clinical implications. A contiguous gene syndrome can be detected by both cytogenetic and molecular analysis using STS and flanking markers as probes (see Chap. 65). In the absence of a contiguous gene syndrome, the rationale for prenatal diagnosis in simple, uncomplicated STS deficiency (X-linked ichthyosis) should be carefully considered with at-risk families from a medical and ethical standpoint. The overall severity and impact on quality of life is usually minimal and can now be quite effectively managed. It is therefore important to be sure that parents have an adequate understanding of the nature of this dermatologic disorder before proceeding with prenatal studies. In addition, experimental models for corrective STS gene transfer into patients' keratinocytes have been developed (see above).189,190
Treatment of X-linked ichthyosis is now quite satisfactory. Lac-Hydrin (12 percent ammonium lactate) is a well-tolerated and effective keratolytic agent that gives good cosmetic results if applied once or twice daily. Older preparations seemed to be associated with unacceptable stinging and itching. Use of cis-retinoic acid is probably contraindicated, although it is useful in the treatment of some other forms of ichthyosis. The risks of teratogenesis are not of direct concern in the male patients under treatment, although reports of hypertriglyceridemia are of note. Furthermore, patients with X-linked ichthyosis seem not to respond to this treatment and may even become worse. In any event, the simple application of a topical keratolytic agent is quite effective and without apparent side effects. The only other point worthy of mention is the possible increased risk of testicular neoplasms in STS-deficient patients. Although the data supporting this association are not compelling at present, it may be appropriate to instruct patients in testicular self-examination and to employ other efforts at prospective diagnosis without unduly alarming these subjects. Because the general health of STS-deficient patients is otherwise good, no other specific management is indicated.