Extensive clinical, hormonal and genetic screening in a large consecutive series of 46,XY neonates and infants with atypical sexual development

Abstract

Background: One in 4500 children is born with ambiguous genitalia, milder phenotypes occur in one in 300 newborns. Conventional time-consuming hormonal and genetic work-up provides a genetic diagnosis in around 20-40% of 46,XY cases with ambiguous genitalia. All others remain without a definitive diagnosis. The investigation of milder cases, as suggested by recent reports remains controversial.

Methods: Integrated clinical, hormonal and genetic screening was performed in a sequential series of 46, XY children, sex-assigned male, who were referred to our pediatric endocrine service for atypical genitalia (2007-2013).

Results: A consecutive cohort of undervirilized 46,XY children with external masculinization score (EMS) 2-12, was extensively investigated. In four patients, a clinical diagnosis of Kallmann syndrome or Mowat-Wilson syndrome was made and genetically supported in 2/3 and 1/1 cases respectively. Hormonal data were suggestive of a (dihydro)testosterone biosynthesis disorder in four cases, however no HSD17B3 or SRD5A2 mutations were found. Array-CGH revealed a causal structural variation in 2/6 syndromic patients. In addition, three novel NR5A1 mutations were found in non-syndromic patients. Interestingly, one mutation was present in a fertile male, underlining the inter- and intrafamilial phenotypic variability of NR5A1-associated phenotypes. No AR, SRY or WT1 mutations were identified.

Conclusion: Overall, a genetic diagnosis could be established in 19% of non-syndromic and 33% of syndromic cases. There is no difference in diagnostic yield between patients with more or less pronounced phenotypes, as expressed by the external masculinisation score (EMS). The clinical utility of array-CGH is high in syndromic cases. Finally, a sequential gene-by-gene approach is time-consuming, expensive and inefficient. Given the low yield and high expense of Sanger sequencing, we anticipate that massively parallel sequencing of gene panels and whole exome sequencing hold promise for genetic diagnosis of 46,XY DSD boys with an undervirilized phenotype.

Figure 1

Mowat-Wilson syndrome, facial characteristics. The typical large and uplifted earlobes in Patient 30, who was diagnosed with Mowat-Wilson syndrome based on clinical data.

Figure 2

Three novel NR5A1 mutations. (A) Schematic overview of the positions of the mutations and electropherograms. (B) RT-qPCR showed a lower NR5A1 expression in the maternal grandfather of the index patient (I:1), and in the mother of the index patient (II:2). We did not include the index case in this experiment as no fresh blood could be collected. Two negative control samples (NC) without the mutation were included for comparison. To exclude technical variations, expression of the reference genes GADPH, HMBS and TBP were also measured, showing stable expression in all patients. (C) Pedigrees for the patients with a NR5A1 mutation. The genotype of the analysed individuals is shown under their symbol. Full black squares indicate affected males with hypospadias, partially black circles indicate females with POF and circles with a black dot correspond with asymptomatic carrier females.

Figure 3

Overview of the integrated investigation approach. (A) Results in the 46,XY undervirilization cohort. Clinical and hormonal investigation was sufficient to suspect a diagnosis in 4/32 cases. For two Kallmann syndrome patients the diagnosis was genetically confirmed, as shown in the CNV analysis and targeted resequencing boxes. A ZEB2 mutation was identified in the Mowat-Wilson syndrome patient. Subsequently a genetic work-up was performed for the remaining patients, guided by hormonal results. Sequencing of HSD17B3 and SRD5A2 in patients with a possible testosterone biosynthesis disorder did not reveal mutations. Genetic screening consisting of array-CGH, DSD MLPA and sequential gene-by-gene sequencing led to the identification of two causal CNVs (of which one KS, see above) and three novel NR5A1 mutations, respectively. (B) Suggested clinical algorithm for the investigation of 46,XY male neonates or infants referred for atypical genitalia. Upper section (orange): clinical investigation, including pregnancy history, medical history and physical examination, enables categorization in cases with and without syndromic features. . Mid-section (blue): In all cases, clinical investigation should be followed by a hormonal work-up, which in turn can be suggestive of gonadal dysgenesis (GD), disorders of the steroid hormone biosynthesis pathway and/or rare forms of CAH (*:Only forms characterized by defective androgen production are implicated here), partial androgen receptor defects or KS. Insights in hormone levels can guide selection of target candidate genes. Lower section (green): After thorough evaluation of clinical and hormonal data, a decision can be made to sequence specific gene panels or to proceed to clinical whole exome sequencing to identify the underlying molecular cause and thereby support the clinical diagnosis. The boxes between brackets (with squared filling) represent single gene tests which can be replaced be the aforementioned gene panels In cases with syndromic features, array-CGH is still a recommended method to identify CNVs.