Genetic diagnosis in XY disorders of sex development

Case 1

A 2-month-old baby was brought to the pediatric outpatient with the concern of atypical genitalia. The gender assigned at birth was male. The parents felt that the baby’s phallus was very small, and he passed urine from an opening located at the base of the phallus. The baby was the first offspring of a non-consanguineously married couple, with a birth weight of 2.7 kg and an uneventful perinatal course. He had been feeding well and gaining weight appropriately. There was no history of recurrent vomiting, progressive hyperpigmentation, or a history of ambiguous genitalia or infertility in the family. On examination, vital parameters and anthropometry were normal, with no signs of dehydration or hyperpigmentation, and normal systemic examination. Scrotum was well-formed and rugose with intrascrotal gonads, and phallic length was 1 cm with chordee and penoscrotal hypospadias. Preliminary investigations yielded normal electrolytes and glucose, cortisol 17.1 µg/dL (reference 2.8–23), luteinizing hormone (LH) 8.4 IU/L (reference range 0.6–4.1), follicle-stimulating hormone (FSH) 4.4 IU/L (reference range 0.4–3.0), 17α-hydroxyprogesterone (17OHP) <0.05 ng/mL (Reference 0.11–1.7), testosterone 12 ng/dL (reference range 20–219), and anti-Müllerian hormone (AMH) 120 ng/mL (reference range 55–210). There were no müllerian structures on ultrasonography. Karyotype was 46,XY. Human chorionic gonadotropin (hCG) stimulation test was done, on which serum testosterone showed an inadequate rise to 59 ng/dL (reference >100) with a testosterone to androstenedione ratio of 0.5 (reference >0.8). The overall clinical picture (palpable gonads; absent Müllerian structures on ultrasonography; normal blood pressure, serum electrolytes, glucose, and cortisol; with low baseline and stimulated testosterone; and normal AMH) was in keeping with a diagnosis of testosterone biosynthetic defect without adrenal insufficiency, specifically 17β-hydroxysteroid dehydrogenase-3 deficiency.

The baby was prescribed three doses of Inj. testosterone 25 mg intramuscularly (IM) at monthly intervals, and subsequently underwent uneventful chordee correction and urethroplasty at the ages of 1 year and 2.5 years, respectively. At the age of 2 years and 8 months, however, he was brought to the pediatric emergency with fever, diarrhea, vomiting, and seizures. On examination, he appeared dehydrated and hyperpigmented. Investigations revealed sodium of 123 mEq/L, potassium 6.7 mEq/L, random blood glucose 25 mg/dL, cortisol 3.3 µg/dL, adrenocorticotropic hormone (ACTH) 1250 pg/mL (reference range 15–65), and 17OHP 0.3 ng/mL (Reference range 0.11–1.7). The diagnosis was revised to a salt wasting form of CAH such as 3β-hydroxysteroid dehydrogenase-2 deficiency, steroidogenic acute regulatory (StAR) defect, or side chain cleavage (CYP11A1) defect [Figure 1]. Dehydroepiandrosterone sulfate (DHEAS) was 0.46 µg/dL (reference 7–126). Exome sequencing identified the following [Box 1]:

A diagnosis of XY DSD and primary adrenal insufficiency due to StAR defect was made.

Case 2

A 2-month-old baby was brought to the pediatric outpatient with the chief complaint of atypical genitalia. The gender assigned at birth was male. Birth, development, and family history were non-contributory. On examination, the left testis was palpable in the inguinal region, while the right testis could not be palpated, and there was micropenis with a phallic length of 1.5 cm, chordee, and penoscrotal hypospadias (external masculinization score 4/12). The rest of the general physical and systemic examination was normal. On evaluation, the baby had a karyotype of 46,XY. Sonogram of the pelvis identified a uterus and both gonads in the inguinal canals. Biochemical investigations were as follows: LH 7.3 IU/L (reference range 0.6–4.1), FSH 6.7 IU/L (reference range 0.4–3.0), testosterone 214 ng/dL (reference range 20–219), and AMH 15.6 ng/mL (reference range 55–210). A diagnosis of XY DSD due to partial gonadal dysgenesis was made. Urine routine examination and ultrasonography of kidneys were normal. The molecular genetic testing was reported as follows [>Box 2]:

A provisional diagnosis of Denys–Drash syndrome was made. A surveillance plan to monitor for the development of Wilms’ tumor and nephrotic syndrome was devised, and parents were counseled about this. After 2 months, the mother presented to us with the concern of having noted an abdominal mass, which was identified on the sonogram as a well-circumscribed hypoechoic mass measuring 6.5 × 6.3 cm and arising from the lower pole of the left kidney. Detailed workup did not identify any local invasion or metastasis. A diagnosis of Wilms’ tumor was made, and the baby received neoadjuvant chemotherapy and underwent a left radical nephrectomy. He has been doing well on follow-up, with the plan to complete his course of adjuvant chemotherapy and monitor for the development of nephrotic syndrome or Wilms’ tumor in the contralateral kidney.

Disorders of gonadal development

Defects in genes that participate in normal testicular development can result in complete or partial gonadal dysgenesis.^[4] Figure 2 depicts the pathway of gonadal development in males and females. Discussing briefly, the bipotential gonad develops from the embryonic mesoderm at about 4 weeks from conception. The development is mediated and sustained by genes such as Wilms’ tumor 1 (WT1), nuclear receptor subfamily 5 group A member 1 (NR5A1), and Wingless-type MMTV integration site family member 4 (WNT4). In fetuses with a Y chromosome, the sex-determining region Y (SRY) gene is activated by WT1, mitogen-activated kinase kinase kinase 1 (MAP3K1), GATA binding protein 4 (GATA4), and Chromobox 2 (CBX2). The protein product of SRY, along with the products of other genes such as NR5A1, WT1, and SRY-box transcription factor 8 (SOX8), in turn, increases the transcription of SRYbox transcription factor 9 (SOX9) by interacting with its upstream enhancer. SOX9 protein causes differentiation of pre-Sertoli cells into Sertoli cells and enhances transcription of fibroblast growth factor 9, which inhibits pathways that lead to ovarian development. Sertoli cells secrete AMH and desert hedgehog (DHH) homolog. AMH causes regression of müllerian structures such that fallopian tubes, uterus, cervix, and the upper one-third of the vagina are not formed, while DHH, along with SF1 (the protein product of NR5A1) and the protein product of Mastermind-like domain containing 1 (MAMLD1), promotes the development of Leydig cells. Leydig cells secrete testosterone and insulin-like peptide-3, both of which participate in testicular descent. A number of testes determining transcription factors encoded by genes such as SOX9, Doublesex, and mab-3 related transcription factor 1 (DMRT1) and Zing ring finger 3 (ZNRF3) repress ovarian development pathways that involve protein products of R-spondin1 (RSPO1), WNT4, Catenin-beta 1 (CTNNB1), Forkhead box L2 (FOXL2), and DAX1 (NR0B1).^[5] It is worthwhile to note that the majority of the sex-determining genes are dosage-sensitive. Insufficiency of the pro-testes genes (WT1, NR5A1, GATA4, MAP3K1, CBX2, SOX9, SOX8, SRY, DHH, DMRT1, ZNRF3, and MAMLD1) and/or excess of the pro-ovarian genes (DAX1, WNT4, FOXL2, RSPO1, and CTNNB1) can cause impaired gonadal development and gonadal dysgenesis in individuals with XY karyotype.

Some of these molecular defects present with isolated gonadal dysgenesis, while others may have syndromic presentation, as they regulate the development of other organ systems as well. Key examples of the latter include Frasier syndrome (focal segmental glomerulosclerosis and gonadoblastoma) and Denys–Drash syndrome (diffuse mesangial sclerosis and Wilms’ tumor) due to defects in WT1 gene, congenital heart disease in GATA4 defects, primary adrenal insufficiency in NR5A1 defects, campomelic dysplasia in SOX9 defects, and polyneuropathy in DHH defects. Again, the presentation is variable in these, with the spectrum of presentations evolving over time as more and more patients are offered molecular testing. In individuals with XY DSD due to WT1 defects, up to 85% of individuals have renal pathology in the form of either early- or late-onset glomerulopathy or Wilms’ tumor.^[6] For some of the variants, the genotype-phenotype correlation has been very strongly established. For example, missense variants in exons 8 and 9 of WT1 are associated with Denys– Drash syndrome, while splice site variants in intron 9 result in Frasier syndrome.^[6,7] In defects of the NR5A1 (formerly SF1) gene, which has emerged as one of the commoner causes of XY DSD, the majority of patients present with a phenotype of partial gonadal dysgenesis with no adrenal insufficiency.

Disorders of gonadal development arise secondary to both single-nucleotide variants (SNVs), which are amenable to detection by gene sequencing-based methods (single gene and panel testing, whole exome sequencing [WES]), as well as copy number variants (CNVs), i.e., deletions or duplications, that are amenable to detection by chromosomal microarray (CMA)-based methods. Certain genes are described to harbor only SNVs, others are described to harbor only CNVs, and yet others are described to harbor a combination of both.^[8]

Disorders of androgen biosynthesis

The classical pathway of androgen biosynthesis starts with cholesterol and cruises through a number of steps before culminating in the production of androgens [Figure 1]. Leydig cells produce testosterone, which is activated by 5α-reductase type 2 to dihydrotestosterone (DHT) in target tissues. Both testosterone and DHT act on AR and result in masculinization of the internal and external genitalia. Besides the classical pathway of androgen biosynthesis, an alternative pathway, also known as the “backdoor” pathway, operates for the synthesis of DHT from cholesterol that does not involve DHEA, androstenedione, and testosterone. In the backdoor pathway, 5α-reductase type 1 (SRD5A1) acts on 17OHP and progesterone. In addition, other enzymes such as aldo-keto reductases (AKR1C2/4), 17β-HSD5 (AKR1C3), and 17β-HSD6 (also called retinol dehydrogenase, RoDH) (HSD17B6) are critical mediators of this pathway. The backdoor pathway plays a crucial role in early male sexual development and physiological male mini-puberty during infancy. Defects in the genes AKR1C2/AKR1C4 result in XY DSD.^[9] Androgen biosynthetic defects are inherited in an autosomal recessive fashion and arise as a result of SNVs that are amenable to genetic diagnosis by sequencing-based methods.^[8] Based on their location in the classical pathway of steroid biosynthesis, the defects may present as isolated DSD (CYB5A, HSD17B3, 5α reductase [SRD5A2], and non-classical form of HSD3B2 defect), as DSD with combined mineralocorticoid and glucocorticoid insufficiency (StAR, CYP11A1, and HSD3B2), or as DSD with mineralocorticoid excess and glucocorticoid insufficiency (CYP17A1, and POR)^[10,11] [Figure 1]. Defects in LH/choriogonadotropin receptor (LHCGR), a gene encoding receptor for both LH and hCG, cause impaired downstream signaling inside Leydig cells and result in reduced androgen synthesis.

Disorders of androgen action

Androgen action defect due to variation in the AR gene, located on the X chromosome, may occur due to both SNVs and CNVs (chiefly deletions).

Disorders of hypothalamic-pituitary-gonadal axis

Hypogonadotropic hypogonadism, identified chiefly in children presenting with delayed puberty, may also manifest in the neonatal period as genital ambiguity with micropenis and bilateral cryptorchidism. It occurs due to defects in genes that participate in development and migration of gonadotropin-releasing hormone (GnRH) neurons (Kallmann syndrome 1 sequence, Fibroblast growth factor 8, Fibroblast growth factor receptor 1, Prokineticin 2, Prokineticin receptor 2, and Chromodomain helicase DNA binding protein 7), GnRH secretion (KiSS-1 metastasis suppressor, Kisspeptin receptor, Tachykinin precursor 3, and Tachykinin receptor 3), and GnRH action (Gonadotropin-releasing hormone receptor).^[12] Up to 60% of patients with idiopathic hypogonadotropic hypogonadism, chiefly due to defective migration of GnRH neurons, also have associated anosmia/hyposmia resulting in Kallmann syndrome. In general, the defects of the hypothalamic-pituitary-gonadal axis occur due to SNVs and can be identified using sequencing-based methods.^[8]

Table 1 lists the genes implicated in various etiologies of XY DSD.

Imaging

Imaging helps to localize gonads and assess the anatomy of the internal genitalia. The presence of testes and the absence of müllerian derivatives point to XY DSD due to androgen synthesis/action defect, whereas the presence/absence of gonads and the presence of müllerian derivatives point to XY DSD due to gonadal dysgenesis. Ultrasonography is the first line of imaging investigation, supplemented by a genitogram and/or magnetic resonance imaging (MRI) of the abdomen and pelvis, as needed.

Hormonal evaluation

A low level of testosterone during mini-puberty or puberty suggests either gonadal dysgenesis or biosynthetic defect. Serum AMH, a marker of immature Sertoli cell function, helps to distinguish isolated biosynthetic defect (normal AMH) from gonadal dysgenesis (low AMH). A normal AMH level is indicative of a functioning testicular tissue and is useful in the evaluation of patients with bilateral cryptorchidism, ovotestes, and XX testicular DSD. AMH level starts to rise soon after birth as Sertoli cells begin to increase, reaches a peak at 6 months of age, and gradually declines through childhood, becoming low during puberty.^[15]

In addition to normal AMH and low testosterone, a low testosterone to androstenedione ratio (cut off of 0.8) is also suggestive of testosterone biosynthetic defect. When a biosynthetic defect is suspected, further evaluation is done to assess if the defect involves the glucocorticoid and/or mineralocorticoid pathways as well by measuring levels of plasma glucose, serum electrolytes (Na⁺/K⁺), serum cortisol, plasma ACTH, and when required, a steroid panel comprising 17OHP, 11-deoxycorticosterone, and DHEAS. When the suspicion of adrenal insufficiency is high with non-specific findings at baseline, dynamic testing using standard dose synacthen (ACTH) stimulation may be required.

If testosterone levels during mini-puberty or puberty are normal to high, 5α-reductase deficiency and androgen insensitivity syndrome (AIS) remain the chief possibilities. The testosterone to DHT ratio is commonly employed to differentiate between the two (high [>20] in 5α-reductase deficiency and low [<20] in AIS). However, the test lacks specificity, with elevated ratios noted in a major proportion of individuals with AIS.^[16]

Evaluation of a prepubertal child with low levels of LH and FSH is challenging because quiescence of the hypothalamic-pituitary-gonadal axis results in investigations that are non-informative. This warrants activation of the androgen biosynthetic pathway by administering hCG, which is also an agonist at the receptor for LH (LHCG receptor). The usual protocol comprises IM administration of Inj. hCG 500–1500 IU/m² every 48 h with an assessment of steroid profile 24 h after the last dose, but the protocol may have to be extended for up to 3 weeks in prolonged hCG testing. The latter is usually done when levels of both testosterone and its precursors are low after a standard hCG stimulation test.^[17]

Surgery

Diagnostic laparoscopy is done if the gonads and/or Müllerian structures cannot be localized on routine imaging. It is typically combined with gonadal biopsy and/or gonadopexy.

Genetic studies

Karyotyping is an essential investigation in children with DSD. Other modalities, such as fluorescent in situ hybridization (FISH) and quantitative fluorescent–polymerase chain reaction (QF-PCR) to identify Y chromosome material, may be used in the interim due to their faster turnaround time. This is especially helpful in an infant with atypical genitalia in whom the diagnostic process needs to be expedited for sex assignment and alleviation of parental anxiety.

Establishing a precise etiology

Molecular diagnosis ends the diagnostic odyssey for the family and the clinician. For the patient and their family, it leads to an acceptance of the diagnosis, brings about an understanding of “why this happened,” and thus helps to ease feelings of stigma, guilt, shame, and blame that often accompany a diagnosis of DSD. More importantly, it helps in bringing out the entity from under the blanket or umbrella term of DSD and gives an exact diagnosis to the child for the under or over-virilization, akin to other endocrinological conditions.

Predicting disease course/trajectory

A molecular diagnosis informs the clinician of hormone production and responsiveness, which can help in choosing appropriate androgen replacement therapy. For example, children with testosterone biosynthetic defects respond well to testosterone, whereas those with 5α-reductase type 2 deficiency and partial AIS (PAIS) benefit from the application of DHT. Molecular diagnosis also provides guidance regarding the most appropriate sex of rearing based on the data available in the literature on pubertal development, adult gender identity, sexual function, and fertility. However, as of now, such information is scant for several molecular etiologies, barring the most common ones, such as AIS, SRD5A2 defect, and HSD17B3 defect for which Sanger sequencing has been in use for some time. This literature is also mostly from Europe and the Middle East. With greater availability of molecular diagnoses using next-generation sequencing (NGS), the phenotypic spectrum, as well as the natural course of several molecular etiologies, will now attain greater clarity and will have the potential to guide future clinicians and families on optimum care.

Prediction of extragenital features

As highlighted in the section on types of XY DSD, a genetic diagnosis is key to predicting the onset of extragenital features. This enables putting in place a surveillance plan for the patient, which can potentially avert life-threatening complications such as adrenal insufficiency (NR5A1, DAX1, StAR, CYP11A1, HSD3B2, and CYP17A1) and glomerulopathy or Wilms’ tumor (WT1).

Reproductive planning for the family

As with all genetic diseases, a molecular diagnosis informs risks of recurrence in other members of the family based on the mode of disease inheritance. This includes timely antenatal counseling and diagnosis, with pregnancy termination decisions backed by parents’ wishes and the severity of the disease in question.

Karyotype

Karyotype is a conventional cytogenetic technique that provides a visual representation of chromosome complement after pre-treated chromosomes are subjected to condensation and Giemsa staining (G-banding). It provides information on alteration in chromosome number and structure provided that the changes are larger than 5,000–10,000 Kb in size. The technique requires that cells are cultured and arrested in metaphase, which is the most appropriate stage for visualizing and studying chromosomes in their most compact state. Guidelines recommend that a minimum of two metaphases must be thoroughly analyzed, although, in practice, many more cells are usually counted.^[18] Due to the requirement of culture of fresh viable cells, the turnaround time ranges from 7 to 10 days.^[19] Karyotype is usually the first genetic investigation that helps to classify DSD based on the underlying chromosomal constitution.

FISH

FISH is also a cytogenetic technique, but unlike karyotype, it has a much higher resolution of 50–500 Kb and a more rapid turnaround time of 24–48 h. It utilizes fluorescent probes directed against specific chromosomal regions to enable the identification of insertions, deletions, and translocations. Its utility in the context of DSD lies in the identification of the Y chromosome/SRY gene. Guidelines recommend the analysis of at least 50 interphase cells for reporting.^[18,20] However, up to 600–2000 interphase cells can be analyzed in cases with a high index of suspicion of sex chromosomal DSD. Thus, FISH is a useful tool for quick identification of chromosomal sex (XX vs. XY), for detecting SRY gene translocation (as in 46, XX testicular or ovotesticular DSD), and for detection of mosaicism when the karyotype is 46,XX, or 45,X.

QF-PCR

QF-PCR is the most rapid method which relies on PCR-based amplification of sex chromosomes. The turnaround time is a few hours, and the results are available at the end of one working day. Among all modalities to identify sex chromosomes, QF-PCR is the fastest and most cost-effective. However, it has limited capabilities for the assessment of mosaicism and partial monosomies.^[8,19]

Single gene testing

Testing of a single gene using Sanger sequencing is undertaken to pinpoint the exact molecular etiology. It is the preferred method when the patient’s phenotype and biochemical investigations are highly suggestive of an underlying genetic diagnosis, as is the case with steroid biosynthetic defects. Sanger sequencing becomes even more attractive if the concerned gene is either small in size or has known hotspots. For example, the SRD5A2 gene is small with 5 exons and has a well-described hotspot (p.R246Q) in the Indian subcontinent.^[16,21] When deletion/duplication of a gene or its segment is a major disease mechanism, for example, in AIS, the test of choice is multiplex ligation-dependent-probe amplification (MLPA). MLPA is a kit-based test, with the kit being specific for particular genes, that is, AR in this case. The overall yield of single gene testing has been found to be poor (~10%), primarily due to significant phenotypic overlap among various disease entities.^[22] However, it must be borne in mind that appropriate patient population selection is a major determinant of the diagnostic rate. For example, in a study by our group, 75 children with a high pre-test probability of 5α-reductase deficiency or PAIS were evaluated sequentially by Sanger sequencing of SRD5A2 and AR genes. Pathogenic/likely pathogenic variants in SRD5A2 were identified in 25/75 (33.3%) and in AR in 19/50 (38%).^[16,23]

Testing for DSD panels

Over the past decade, advances in molecular techniques have led to the development of DSD panels comprising different numbers of DSD-related genes. Genes in such panels are sequenced using massive parallel high throughput sequencing techniques (e.g., NGS) to enable the identification of SNVs and small insertions and deletions (indels) in protein-coding regions of DSD-related genes. A diagnostic yield ranging from 31% to 46% has been obtained from studies utilizing DSD panels comprising 30–219 genes in patients with XY DSD.^[22,24-29]

In different series, including ours, SRD5A2, AR, and NR5A1 have been the most frequently implicated genes in 46,XY DSD.^{[16,22,24-28,30,31]}

CMA

CMA is used to identify microdeletions and microduplications well beyond the threshold of conventional cytogenomic techniques. At a resolution of 10 Kb, it can identify small intragenic as well as large structural CNVs. As opposed to MLPA, which identifies CNVs in a single gene, CMA scans the entire genome for deletions and duplications.^[19,32] The two main types of CMA are single-nucleotide polymorphism array and array comparative genomic hybridization. The utility of this modality in the context of XY DSD lies mainly in the diagnosis of cases of gonadal dysgenesis, with an overall yield of ~30%; 5.6% in isolated gonadal dysgenesis, increasing all the way up to 25% in syndromic gonadal dysgenesis.^[33] Etiologies such as DAX1 deletion (resulting in hypogonadotropic hypogonadism and adrenal hypoplasia congenita), DAX1 duplication (resulting in XY complete gonadal dysgenesis), and a deletion upstream of SOX9 in its regulatory region (resulting in XY gonadal dysgenesis) may be identified using CMA.^[34] CMA has conventionally been an underutilized technique, with recent studies showing progressively greater yield ^[35,36] and recent guidelines placing it higher up in the diagnostic algorithm for the evaluation of DSD.^[8,14,37-40]

WES

WES captures a nucleic acid sequence of the entire protein-coding region of the genome using high throughput sequencing technologies. The raw data are initially screened for relevant DSD genes and their immediately adjacent splice sites. In case no disease-causing variant is identified, the analysis proceeds to include other genes. Thus, WES has facilitated the discovery of novel genes and unraveled new phenotypes associated with previously reported genes (e.g., NR5A1).^[34,40-43] WES has also brought into focus the phenomenon of oligogenic inheritance, wherein hits in multiple genes involved in the pathway to determine the final phenotype may culminate in the structural and functional manifestation in an individual. In XY DSD, oligogenic inheritance has been postulated for NR5A1, MAP3K1, AR, HSD3B2, WT1, WDR11, GATA4, DHH, etc.^[44]

It is generally preferred to do WES in trios comprising the index patient and both parents. This increases the yield of testing as it allows several variants of uncertain significance (VUS) to be upgraded based on their presence or absence in symptomatic or asymptomatic parents.^[45] However, this is not necessarily the case with DSDs, where the expression of many genes is sex-restricted.^[28] For example, a boy with XY gonadal dysgenesis and a heterozygous variant in the NR5A1 gene may have inherited the variant from his asymptomatic mother, who may either continue to remain asymptomatic or go on to develop premature ovarian failure at a later age (not manifest at the time of testing). The major limitations of WES include the identification of VUS, detection of incidental findings unrelated to the phenotype that may pose significant ethical dilemmas, exclusion of non-coding regions, and inability to identify larger structural variants.

Whole genome sequencing (WGS)

Currently confined to the research setting, WGS is a powerful tool capable of sequencing the entirety of the human genome with accuracy and identifying both SNVs and CNVs in coding as well as non-coding regions of the genome. It is predicted to result in an impressive diagnostic yield for all genetic disorders. WGS comes with several limitations, though, the chief ones being its exorbitant cost, and enormous volume of data with increased cost of storage, increased complexity of analysis, and limited expertise in interpretation.^[8]

Summary of genetic testing

Despite the armamentarium of currently available genetic tests, the choice of investigation is of utmost importance in obtaining a quick and accurate diagnosis and reducing the financial burden on patients’ families. The role of a focused clinical history and examination, appropriate baseline hormonal assays (especially when the patient presents in mini-puberty or puberty), and ultrasonography cannot be overemphasized. However, genetic testing can be given precedence over doing dynamic or expensive endocrine testing, and over imaging-based investigations that are invasive or require anesthesia.

When the suspicion of a monogenic etiology is extremely high, single gene testing using Sanger sequencing or MLPA may be undertaken depending on whether the gene is known to be primarily affected by SNVs or CNVs. In the absence of a clear diagnosis, multiple genes may be sequenced simultaneously (panel testing via NGS) or subject to CMA; with a lower threshold to opt for CMA in cases of multiple congenital malformations, developmental delay, and intellectual disability.^[19] When a structured approach is adopted, the yield of genetic testing in XY DSD has been seen to improve significantly to 64%.^[30] Ideally, before and after ordering any genetic test, and especially when ordering an exome or genome level test, in-depth pre- and post-test counseling sessions need to be organized with the clinical geneticist and the families, with provision of information on expectations from the genetic test; its cost, turnaround time, and fallacies; the need to verify the diagnosis with further testing such as parental segregation; the interpretation of the test result; and the usefulness of the test in predicting disease trajectory and in reproductive planning.^[1,39,46]

Fallacies of genetic testing

The shortcomings of genetic testing in children with DSD can be broadly grouped under the following heads: