A Survey of Compound Heterozygous Variants in Pediatric Cancers and Structural Birth Defects

Compound heterozygous (CH) variants occur when two recessive alleles are inherited and the variants are located at different loci within the same gene in a given individual. CH variants are important contributors to many different types of recessively inherited diseases. However, many studies overlook CH variants because identification of this type of variant requires knowing the parent of origin for each nucleotide. Using computational methods, haplotypes can be inferred using a process called “phasing,” which estimates the chromosomal origin of most nucleotides. In this paper, we used germline, phased, whole-genome sequencing (WGS) data to identify CH variants across seven pediatric diseases (adolescent idiopathic scoliosis: n = 16, congenital heart defects: n = 709, disorders of sex development: n = 79, ewing sarcoma: n = 287, neuroblastoma: n = 259, orofacial cleft: n = 107, and syndromic cranial dysinnervation: n = 172), available as parent-child trios in the Gabriella Miller Kids First Data Resource Center. Relatively little is understood about the genetic underpinnings of these diseases. We classified CH variants as “potentially damaging” based on minor allele frequencies (MAF), Combined Annotation Dependent Depletion scores, variant impact on transcription or translation, and gene-level frequencies in the disease group compared to a healthy population. For comparison, we also identified homozygous alternate (HA) variants, which affect both gene copies at a single locus; HA variants represent an alternative mechanism of recessive disease development and do not require phasing. Across all diseases, 2.6% of the samples had a potentially damaging CH variant and 16.2% had a potentially damaging HA variant. Of these samples with potentially damaging variants, the average number of genes per sample was 1 with a CH variant and 1.25 with a HA variant. Across all samples, 5.1 genes per disease had a CH variant, while 35.6 genes per disease had a HA variant; on average, only 4.3% of these variants affected common genes. Therefore, when seeking to identify potentially damaging variants of a putatively recessive disease, CH variants should be considered as potential contributors to disease development. If CH variants are excluded from analysis, important candidate genes may be overlooked.

Download Full-text

Characterising SRD5A2 Gene Variants in 37 Indonesian Patients with 5-Alpha-Reductase Type 2 Deficiency

International Journal of Endocrinology ◽

10.1155/2019/7676341 ◽

2019 ◽

Vol 2019 ◽

pp. 1-10 ◽

Cited By ~ 1

Author(s):

Nanis S. Marzuki ◽

Firman P. Idris ◽

Hannie D. Kartapradja ◽

Alida R. Harahap ◽

Jose R. L. Batubara

Keyword(s):

Autosomal Recessive ◽

Disorders Of Sex Development ◽

External Genitalia ◽

Compound Heterozygous ◽

Phenotypic Characteristics ◽

Sex Development ◽

Srd5a2 Gene ◽

Diagnostic Approaches ◽

Autosomal Recessive Condition

The 5-alpha-reductase type 2 deficiency (5ARD2) is an autosomal recessive condition associated with impairment in the conversion of testosterone to dihydrotestosterone. This condition leads to undervirilisation in 46,XY individuals. To date, there have been more than 100 variations identified in the gene responsible for 5ARD2 development (steroid 5-alpha-reductase 2, SRD5A2). However, few studies have examined the molecular characterisation of Indonesian 5ARD2 cases. In the current study, we analysed 37 subjects diagnosed with 46,XY DSD (disorders of sex development) with confirmed variations in the SRD5A2 gene. We examined results from testosterone/dihydrotestosterone (T/DHT) and urinary etiocholanolone/androsterone (Et/An) ratios, as well as from molecular and clinical analyses. Twelve variants in the SRD5A2 gene were identified, and 6 of which were novel, namely, c.34–38delGinsCCAGC, p.Arg50His, p.Tyr136∗, p.Gly191Arg, p.Phe194Ile, and p.Ile253Val variants. Moreover, we determined that 20 individuals contained harmful mutations, while the remaining 17 variants were benign. Those containing harmful mutations exhibited more severe phenotypes with median external genitalia masculinisation scores (EMS) of 3 (1.5–9) and were more likely to be diagnosed at a later age, reared as female, and virilised at pubertal age. In addition, the respective sensitivities for detecting severe 5ARD2 cases using T/DHT (cutoff: 10) and urinary Et/An ratios (cutoff: 0.95) were 85% and 90%, whereas mild cases were only identified with 64.7% and 47.1% sensitivity, respectively. Although we were unable to identify clear correlations between genotypic and phenotypic characteristics in this study, we clearly showed that individuals who were homozygous or compound heterozygous for any of the harmful mutations were more likely to exhibit classic 5ARD2 phenotypes, lower EMS, female assignment at birth, and virilisation during puberty. These results serve to inform the development of improved clinical and molecular 5ARD2 diagnostic approaches, specifically in Indonesian patients.

Download Full-text

Molecular Characteristics of Sequence Variants in GATA4 in Patients with 46,XY Disorders of Sex Development without Cardiac Defects

Sexual Development ◽

10.1159/000511258 ◽

2019 ◽

Vol 13 (5-6) ◽

pp. 240-245

Author(s):

Jin-Ho Choi ◽

Yena Lee ◽

Arum Oh ◽

Gu-Hwan Kim ◽

Han-Wook Yoo

Keyword(s):

Heart Defects ◽

Disorders Of Sex Development ◽

External Genitalia ◽

Pathogenic Mutation ◽

Luciferase Reporter ◽

Molecular Characteristics ◽

Sequence Variants ◽

Sex Development ◽

Stimulation Tests

A GATA4 haploinsufficiency has been well described in patients with congenital heart defects (CHDs), whilst only a few studies have reported mutations related to a 46,XY disorder of sex development (DSD) phenotype. This study investigated the clinical phenotypes and molecular characteristics of two 46,XY DSD patients harboring GATA4 variants. Mutation analysis was performed using a targeted gene panel or whole-exome sequencing. The transactivation activity of each variant protein was examined by in vitro luciferase reporter assay using the AMH and SRY promoters. Subject 1 presented with a micropenis and hypospadias. Subject 2 showed complete female external genitalia with a 46,XY karyotype. Both patients were responsive to hCG stimulation tests and did not manifest CHD. A novel heterozygous variant, c.643A>G (p.R215G), in GATA4 was identified in Subject 1, whereas Subject 2 harbored a previously reported variant, c.1220C>A (p.P407Q), in GATA4 and a previously known pathogenic mutation, i.e., c.226C>T (p.Q76*) in the AR gene. The reporter assays using the SRY and AMH promoters revealed decreased transcriptional activity of both p.P407Q and p.R215G. However, the GATA4 p.P407Q variant was classified as likely benign. In conclusion, it is essential to integrate clinical features and endocrine findings when interpreting sequence variants.

Download Full-text

Spectrum of Pathogenic Variants in SRD5A2 in Indian Children with 46,XY Disorders of Sex Development and Clinically Suspected Steroid 5α-Reductase 2 Deficiency

Sexual Development ◽

10.1159/000509812 ◽

2019 ◽

Vol 13 (5-6) ◽

pp. 228-239

Author(s):

Anil Kumar ◽

Rajni Sharma ◽

Mohammed Faruq ◽

Varun Suroliya ◽

Manoj Kumar ◽

...

Keyword(s):

Genetic Analysis ◽

Wide Spectrum ◽

Disorders Of Sex Development ◽

Pathogenic Variant ◽

Compound Heterozygous ◽

Phenotype Correlation ◽

Indian Children ◽

Sex Development ◽

Pathogenic Variants ◽

Genotype Phenotype Correlation

The aim of this study was to assess the prevalence of pathogenic variants in the SRD5A2 gene in children with 46,XY disorders of sex development (DSD) with normal to high serum testosterone levels and absence of Müllerian structures on imaging and to evaluate the genotype-phenotype correlation. Seventy-five patients with 46,XY DSD and probable clinical diagnosis of 5α-reductase 2 deficiency or androgen insensitivity syndrome were enrolled. Genetic analysis was done for pathogenic variants in SRD5A2, and the genotype-phenotype correlation was studied. As a result, 10 pathogenic or likely pathogenic biallelic variants in SRD5A2, either homozygous or compound heterozygous, were identified in 25 of 75 (33.3%) patients. The hCG stimulated testosterone: dihydrotestosterone (T:DHT) ratio was elevated in all patients with pathogenic variants in SRD5A2 and in nearly 90% of those without pathogenic variants in SRD5A2 in whom this was assessed. The missense pathogenic variant p.R246Q was a hotspot. One novel pathogenic variant p.Y178*, and a variant p.F194I, not previously reported in patients with 5α-reductase 2 deficiency, were identified. The missense variant p.F194I was predicted as deleterious and damaging by in silico analysis and as likely to reduce the enzyme activity by protein modeling. In conclusion, pathogenic variants in SRD5A2 can be detected in a wide spectrum of Indian patients with 46,XY DSD. Molecular genetic analysis should be considered as a first-line test as the T:DHT ratio lacks specificity and a hotspot variant is present in a vast majority.

Download Full-text

SUN-074 Loss-Of-Function Mutations in GATA4 in Patients with 46,XY Disorders of Sex Development Without Cardiac Defects

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.714 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Yena Lee ◽

Arum Oh ◽

Han-Wook Yoo ◽

Jin-Ho Choi

Keyword(s):

Heart Defects ◽

Disorders Of Sex Development ◽

External Genitalia ◽

Luciferase Reporter ◽

Molecular Characteristics ◽

Incomplete Penetrance ◽

Loss Of Function ◽

Sex Development ◽

Wide Range

Abstract Background: Disorders of sex development (DSD) encompass a wide range of conditions associated with numerous causative genes. In about 50-60% of 46,XY DSD individuals, the underlying molecular cause remains uncertain. GATA4 haploinsufficiency has been described in patients with congenital heart defects (CHD), while only a few studies reported mutations related to 46,XY DSD phenotype. This study investigated clinical phenotypes and molecular characteristics of two 46,XY DSD patients with GATA4 mutations. Methods: Mutation analysis was performed in patients with 46,XY DSD by whole exome sequencing (WES) using Illumina NextSeq platform. Clinical and endocrine characteristics were reviewed retrospectively. GATA4 variants identified by WES were verified by Sanger sequencing. Functional activity of GATA4 variants was tested by luciferase reporter assay on the SRY and AMH promoter using two different cell systems including HEK293 and NCI-H295R. Results: Subject 1 presented with micropenis and hypospadias at the age of 5 months. Karyotype was 46,XY. Mullerian duct remnants were not found in pelvic ultrasound. The patient underwent urethroplasty at the age of 10 months and was reared as a male. Subject 2 with complete female external genitalia was referred to our hospital because of 46,XY karyotype on G-scanning. The patient underwent laparoscopic orchiectomy at the age of 1.8 years and was assigned as a female. Both patients were responsive to hCG stimulation tests and did not have CHD. Subject 1 harbored a novel heterozygous variant of c.643A>G (p.R215G)] in GATA4, whereas a previously reported variant of c.1220C>A (p.P407Q) was identified in Subject 2. In vitro luciferase reporter assays using SRY and AMH promoter revealed decreased transcriptional activity of both p.P407Q and p.R215G. Conclusions: This study expanded phenotypic spectrum of mutations in GATA4 in patients with 46,XY DSD without CHD. GATA4 mutations in patients with 46,XY DSD may not be associated with CHD. Possible explanations for phenotypical variability comprise incomplete penetrance, variable expressivity, and oligogenic mechanisms.

Download Full-text

New insights into 5α-reductase type 2 deficiency based on a multi-centre study: regional distribution and genotype–phenotype profiling of SRD5A2 in 190 Chinese patients

Journal of Medical Genetics ◽

10.1136/jmedgenet-2018-105915 ◽

2019 ◽

Vol 56 (10) ◽

pp. 685-692 ◽

Cited By ~ 2

Author(s):

Baoheng Gui ◽

Yanning Song ◽

Zhe Su ◽

Fei-Hong Luo ◽

Linqi Chen ◽

...

Keyword(s):

Regional Distribution ◽

Clinical Manifestations ◽

Disorders Of Sex Development ◽

Chinese Patients ◽

Compound Heterozygous ◽

Multi Centre Study ◽

Proximal Hypospadias ◽

Sex Development ◽

Distal Hypospadias

BackgroundThe 5α-reductase type 2 (5α-RD2) deficiency caused by mutations in the steroid 5α-reductase 2 (SRD5A2) gene results in variable degrees of undervirilisation in patients with 46,XY disorders of sex development. This study aims to profile the regional distribution and phenotype–genotype characteristics of SRD5A2 in a large Chinese 5α-RD2 deficiency cohort through multi-centre analysis.Methods190 subjects diagnosed with 5α-RD2 deficiency were consecutively enrolled from eight medical centres in China. Their clinical manifestations and genetic variants were analysed.ResultsHypospadias (isolated or combined with microphallus and/or cryptorchidism) was fairly common in the enrolled subjects (66.32%). 42 variants, including 13 novel variants, were identified in SRD5A2. Homozygous and compound heterozygous mutations presented in 38.42% and 61.58% of subjects, respectively, and predominated in exons 1, 4 and 5. The most prevalent variant was c.680G > A (52.37%), followed by c.16C > T, (10.79%), c.607G > A, (9.21%) and c.737G > A, (8.95%). However, their distributions were different: c.680G > A was more common in South China than in North China (62.62% vs 39.16%, p < 0.001), whereas the regional prevalence of c.16C > T was reversed (6.07% vs 16.87%, p = 0.001). Furthermore, c.680G > A prevailed in cases with normal meatus (68.75%) or distal hypospadias (66.28%), compared with those with proximal hypospadias (35.54%, p < 0.001). However, cases with proximal hypospadias showed a higher frequency of c.16C > T (20.48%) than those with normal meatus (3.13%) or distal hypospadias (3.49%, p < 0.001).ConclusionsThis study profiled variable phenotypic presentation and wide mutational spectrum of SRD5A2, revealing its distinctive regional distribution in Chinese patients and further shaping the founder effect and genotype–phenotype correlation of SRD5A2.

Download Full-text