All mutants assessed for residual enzymatic activity of both CYP17A1 catalytic reactions show greatly reduced or even absent activity in vitro (Figure 28). However, three mutants (p.R347H, p.A398E and p.F53/54 del) retain some residual 17α- hydroxylase activity of about 10-15 % of WT activity. Amongst these, the p.347H mutation has the highest residual activity (15.6 %) with p.A398E and p.F53/54del retaining about 10% of WT enzyme activity (Figure 28A). The 17,20 lyase activity was greatly reduced to about 1% or less of WT activity for all mutant CYP17A1 protein studied (Figure 28B). Amongst all mutant proteins, p.P409L, p.G111V and the frameshift mutation most severely reduce CYP17A1 catalytic function for both 17α-hydroxylase and 17,20 lyase activities, with the two latter mutations completely abolishing protein function.
Chapter 5 Phenotypic Variability of 17OHD
___________________________________________________________________
Figure 28: Residual mutant enzyme activity values are depicted for the two catalytic activities of
CYP17A1. Panel A for the conversion of progesterone (Prog) to 17-hydroxyprogesterone (17OHP), reflecting 17α-hydroxylase activity; panel B reflects the 17,20 lyase activity as assessed by the conversion of 17-hydroxypregnenolone (17Preg) to DHEA. Residual enzyme activity is expressed at percentage of wild type (WT) activity, which is defined as 100%. Substrate conversion rate for WT protein was 14.4 ± 0.4 nmol/mg protein/min for Prog and 5.8 ± 1.5 nmol/mg protein/min for 17Preg. All experiments were performed in triplicate in three independent experiments. Error bars represent ± SEM (in percent).
5.4. D
ISCUSSIONWe have described five cases with inactivating mutations in the CYP17A1 gene causing 17α-hydroxylase deficiency and demonstrated in vivo (urinary metabolite analysis) and in vitro (functional characterizations) that residual 17α-hydroxylase activity of distinct CYP17A1 mutations is associated with non-classical manifestations of this disorder.
The complete absence of 17α-hydroxylase activity, as illustrated in case 4, caused by the severe homozygous frameshift mutation, resulted in a severe neonatal presentation with adrenal insufficiency. Neonatal presentation resembling primary adrenal insufficiency is very unusual in 17OHD and has not been reported in the literature so far. The majority of patients present ‘classically’, i.e. with 46,XY DSD or lack of pubertal development in 46,XX girls, hypokalaemic hyporeninaemic
Chapter 5 Phenotypic Variability of 17OHD
___________________________________________________________________ hypertension and compensated glucocorticoid deficiency due to partial activation of the GC receptor by MC precursors (Goldsmith et al., 1967; Yanase, 1995; Miura et al., 1996; Krone and Arlt, 2009; Miller and Auchus, 2011); this phenotype is illustrated by patient 1 of this case series. Some excretion of MC metabolites in the baby (case 1) - as detected in her urine sample - might have prevented her from severe salt-wasting crisis, but was also directional in establishing the diagnosis as other causes of primary adrenal insufficiency had already been considered (i.e. mutations in NR5A1, DAX1 or CYP11A1). Another severe mutation, in a homozygous state, resulting in early truncation of the protein (p.Y27X) was previously reported in an adult woman, who had no signs of pubertal development, and interestingly, did not present with arterial hypertension (Müssig et al., 2005). However, the urinary steroid profile in this patient with high MC metabolites but undetectable 17-oxygenated C21 and C19 steroids represented the typical biochemical fingerprint of severe combined 17α-hydroxylase/17,20 lyase deficiency.
About 10% of patients with 17OHD are normokalaemic and normotensive, despite the fact that they carry severe disease-causing mutations (Kater and Biglieri, 1994; Auchus, 2001). The exact explanation for this phenotypic variability is not known and underlying mechanisms remain to be elucidated.
The p.F53/54del mutation in CYP17A1 has been frequently reported and is one of the most prevalent mutations in patients of Asian origin, usually associated with partially combined inactivation of 17α-hydroxylase/17,20 lyase activities (Miura et al., 1996; Bao et al., 2011; Yanase et al., 1989). The predominant phenotype of patients with this mutation is 1) a variable degree of hyperaldosteronism with hypokalaemic hypertension not always present and 2) milder sex steroid deficiency with absent or mild DSD in 46,XY individuals and compensated hypergonadotrophic hypogonadism
Chapter 5 Phenotypic Variability of 17OHD
___________________________________________________________________ in both sexes (Yanase, 1995; Miura et al., 1996). These features are consistent with the clinical presentations of the two siblings with homozygous p.F53_54del from this case series (case 2 and 3) and the in vitro functional analysis of this mutation retaining about 10% residual activity of 17α-hydroxylase and some residual activity of 17,20 lyase (section 5.3.3) may explain the observed phenotype.
Case 5 presented with the clinical and biochemical features of ‘isolated’ 17,20 lyase deficiency. Isolated 17,20 lyase deficiency is a rare condition and only around 25 cases have been described in the literature so far (Yanase et al., 1991) (see also sections 1.3.1.1 and 6.4). Importantly, the initial reports on the condition provided only the clinical and hormonal characterizations without identifying a distinct genetic abnormality in six families with 46,XY DSD patients and two families with 46,XX patients that presented with lack of pubertal development [reviewed and discussed in (Yanase et al., 1991)]. So far, 15 cases of isolated 17,20 lyase deficiency with a complete clinical, hormonal, genetic, and functional work-up have been reported Underlying causes were four distinct missense mutations in the CYP17A1 gene (p.R347H; p.R347C p.R358Q; p.E305G) (Geller et al., 1997; Sherbet et al., 2003; Van Den Akker et al., 2002) and one POR missense mutation (p.G539R) (Hershkovitz et al., 2008). Recently, a CYB5A nonsense mutation (p.W27X) has been reported resulting in early protein truncation and thus loss of CYB5A function (Kok et al., 2010). Case 5 carried the known missense mutation p.R347H on one allele and a novel missense mutation p.A398E on the other allele. A unifying characteristic of all individuals with 17,20 lyase deficiency is severe sex steroid deficiency, with hormonal measurements confirming a lack of adrenal and gonadal androgen synthesis. However, it is noteworthy that all patients with underlying
Chapter 5 Phenotypic Variability of 17OHD
___________________________________________________________________ glucocorticoid production, with insufficient cortisol responses to ACTH stimulation; a comprehensive overview of all the published cases with a genetic work-up available is shown in Table 17. Of note and to our current knowledge, case 5 is the first patient with isolated 17,20 lyase deficiency due to mutant CYP17A1 who has normal ACTH- stimulated cortisol levels (Table 14), however urinary steroid metabolome analysis demonstrated mild impairment of 17α-hydroxylase activity. A comparison of urinary metabolite ratios reflecting 17α-hydroxylase activities between the patients of this case series shows that this ratio is the lowest in this patient, indicating a higher residual in vivo 17α-hydroxylase activity (Figure 26). In addition, the degree of 17α- hydroxylase inhibition reflected by this ratio mirrors the in vitro functional 17α- hydroxylase activity of the mutant enzymes (Figure 28). A comparison of these ratios reflecting between the cases from this thesis and published cases presenting with either classical combined CYP17A1 deficiencies (Neres et al., 2010) or isolated 17,20 lyase deficiencies (Tiosano et al., 2008) is shown in Figure 29. This figure also depicts the three siblings with isolated 17,20 lyase deficieny due to mutant CYB5A, as described in Chapter 6, and data from the Birmingham cohort of patients with PORD (Krone et al., 2012). Clearly, case 5 (represented by the open diamond) has only a mild elevation the 17α-hydroxylase ratio; however, biochemically, the enzymatic activity is inhibited, although there is no clinical or hormonal evidence for impaired GC production or MC excess.
Chapter 5 Phenotypic Variability of 17OHD
___________________________________________________________________
Figure 29: In vivo assessment of CYP17A1 17α-hydroxylase and 17,20 lyase activities as indicated
by urinary steroid metabolite analysis. Diagnostic steroid metabolite ratios in the three siblings with isolated 17,20 lyase deficiency (ILD) due to homozygous p.H44L CYB5A are represented by closed circles on the left of each panel (white, case 1; black, case 2; grey, case 3). The white diamond symbol represents the ratio obtained from the patient with ILD due to mutant CYP17A1 (case 5 in this chapter; p.A398E/p.R347H) and the three patients with a more classical presentation of 17OHD (circle: case 1, p.G111V/p.P409L; triangle: case 2; square: case 3, both p.F53_54del hom). White box plots represent the interquartile ranges of the reference cohort (healthy males and females, 4-20 years; n=98), whiskers represent the 5th and 95th percentiles, respectively. Grey box plots indicate the ranges and medians for the same steroid ratios measured in 20 patients with classic 17α-hydroxylase deficiency (CYP17A1 17OHD; Neres et al. 2010); six patients with apparently isolated 17,20 lyase deficiency due to CYP17A1 p.E305G (CYP17A1 ILD; Tiosano et al. 2008), and 21 patients with P450 oxidoreductase deficiency (Krone et al. 2012). The triangles represent two patients with the POR mutation p.G539R reported as associated with apparently isolated 17,20 lyase deficiency (Hershkovitz et al., 2008). For steroid abbreviations please see methods.
Thus, urinary steroid profiling represents a powerful diagnostic tool, not only to simplify diagnosis of distinct steroidogenic disorders but also to potentially predict the severity of the mutation, as illustrated in this case series. Importantly, true isolated 17,20 lyase deficiency, i.e. no clinical, hormonal or biochemical evidence of impaired GC or MC biosynthesis and action, is not observed in mutations of the CYP17A1 and