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An exonic splicing enhancer mutation in DUOX2 causes aberrant alternative splicing and severe congenital hypothyroidism in Bama pigs.

ABSTRACT: Pigs share many similarities with humans in terms of anatomy, physiology and genetics, and have long been recognized as important experimental animals in biomedical research. Using an N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we previously identified a large number of pig mutants, which could be further established as human disease models. However, the identification of causative mutations in large animals with great heterogeneity remains a challenging endeavor. Here, we select one pig mutant, showing congenital nude skin and thyroid deficiency in a recessive inheritance pattern. We were able to efficiently map the causative mutation using family-based genome-wide association studies combined with whole-exome sequencing and a small sample size. A loss-of-function variant (c.1226 A>G) that resulted in a highly conserved amino acid substitution (D409G) was identified in the DUOX2 gene. This mutation, located within an exonic splicing enhancer motif, caused aberrant splicing of DUOX2 transcripts and resulted in lower H₂O₂ production, which might cause a severe defect in thyroid hormone production. Our findings suggest that exome sequencing is an efficient way to map causative mutations and that DUOX2 ^D409G/D409G mutant pigs could be a potential large animal model for human congenital hypothyroidism.

SUBMITTER: Cao C

PROVIDER: S-EPMC6361156 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Project description:BackgroundCongenital adrenal hyperplasia (CAH) is a heterogeneous group of adrenal steroidogenesis disorders with variable degrees of glucocorticoid, mineralocorticoid and sex steroid deficiencies. CYP11A1 gene encodes the mitochondrial cholesterol side-chain cleavage enzyme (P450scc), which initiates the first reaction in steroidogenesis by converting cholesterol to pregnenolone. Variants in this gene are extremely rare but associated with severe forms of CAH due to its early and critical function in various steroid biosynthesis pathways. Here, we report a CYP11A1 exonic homozygous variant that, although exonic in location, affects splicing by creating an additional aberrant splicing site with frameshift and truncation of the gene.Patients and methodsThe proband is a 23-year old 46,XY patient raised as a girl. She was a product of normal pregnancy for first-degree relative parents. Soon after birth, she had vomiting, dehydration, hypotension, hyponatremia and hyperkalemia. She was started on glucocorticoids and mineralocorticoids with prompt recovery. Apart from a chronic need for these medications, her neonatal and childhood history was unremarkable. She sought medical advice at age 19 years for delayed puberty with primary amenorrhea and lack of breast development. On evaluation, she had normal external female genitalia, no breast development, undescended testes and absent uterus and ovaries. Her hormonal evaluation revealed very low estrogen, testosterone, cortisol, aldosterone, 17-hydroxyprogesterone, and androstenedione levels. ACTH, LH, FSH and renin were very high consistent with primary gonadal and adrenal failure. Her parents are healthy first-degree cousins. She has three sisters, all with 46,XX karyotype. One of them is clinically and biochemically normal while the other two sisters have normal female phenotype, normal uterus and ovaries, similar hormonal profile to the proband but different karyotype (46,XX) and absence of undescended testes. gDNA was used for whole exome sequencing (WES). Sanger sequencing was performed to confirm the detected variant and its segregation with the disease.ResultsWES identified a homozygous missense variant in CYP11A1 changing the second nucleotide (GCG > GTG) at position 189 in exon 3 and resulting in a change of Alanine to Valine (p.Ala189Val). This variant was confirmed by PCR and Sanger sequencing. It was found in a homozygous form in the proband and her two affected sisters and in a heterozygous form in the unaffected sister. In-silico analysis predicted this variant to create a new splicing site with frameshift and truncation of the gene transcript. This was confirmed by isolation of RNA, cDNA synthesis, gel electrophoresis and sequencing.ConclusionWe describe a family with a very rare form of CAH due to a CYP11A1 variant leading to creation of a new splice site, frameshift and premature truncation of the protein.

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An exonic splicing enhancer mutation in DUOX2 causes aberrant alternative splicing and severe congenital hypothyroidism in Bama pigs.

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