RESUMEN
Mutations in the HSD17B3 gene cause HSD17B3 deficiency and result in 46, XY Disorders of Sex Development (46, XY DSD). The diagnosis of 46, XY DSD is very challenging and not rarely is confirmed only at older ages, when an affected XY female presents with primary amenorrhea or develops progressive virilization. The patient described in this paper represents a case of discrepancies between non-invasive prenatal testing (NIPT) and ultrasound based fetal sex determination detected during prenatal screening. Exome sequencing was performed on the cell free fetal DNA (cffDNA), amniotic fluid, and the parents. Libraries were generated according to the manufacturer's protocols using TruSight One Kits (Illumina Inc., San Diego, CA, USA). Sequencing was carried out on NEXT Seq 500 (Illumina) to mean sequencing depth of at least 100×. A panel of sexual disease genes was used in order to search for a causative variant. The finding of a mutation (c.645 A>T, p.Glu215Asp) in HSD17B3 gene in amniotic fluid as well as in cffDNA and both parents supported the hypothesis of the HSD17B3 deficiency. In conclusion, we used clinical exome sequencing and non-invasive prenatal detection, providing a solution for NIPT of a single-gene disorder. Early genetic diagnoses are useful for patients and clinicians, contribute to clinical knowledge of DSD, and are invaluable for genetic counseling of couples contemplating future pregnancies.
Asunto(s)
Ácidos Nucleicos Libres de Células/genética , Trastorno del Desarrollo Sexual 46,XY/genética , Disgenesia Gonadal 46 XY/genética , Desarrollo Sexual/genética , 17-Hidroxiesteroide Deshidrogenasas/genética , Adulto , Femenino , Pruebas Genéticas/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Homocigoto , Humanos , Mutación/genética , Fenotipo , Embarazo , Diagnóstico Prenatal/métodos , Virilismo/genética , Secuenciación del Exoma/métodos , Adulto JovenRESUMEN
The skeletal muscle ryanodine receptor (RyR1), i.e., the Ca2+ channel of the sarco/endoplasmic reticulum (S/ER), and the voltage-dependent calcium channel Cav1.1 are the principal channels involved in excitation-contraction coupling in skeletal muscle. RYR1 gene variants are linked to distinct skeletal muscle disorders, including malignant hyperthermia susceptibility and central core disease (CCD), mainly with autosomal dominant inheritance, and autosomal recessive myopathies with a broad phenotypic and histopathological spectrum. The age at onset of RYR1-related myopathies varies from infancy to adulthood. We report the identification of four RYR1 variants in two Italian families: one with myopathy and variants c.4003C>T (p.R1335C) and c.7035C>A (p.S2345R), and another with CCD and variants c.9293G>T (p.S3098I) and c.14771_14772insTAGACAGGGTGTTGCTCTGTTGCCCTTCTT (p.F4924_V4925insRQGVALLPFF). We demonstrate that, in patient-specific lymphoblastoid cells, the c.4003C>T (p.R1335C) variant is not expressed and the in-frame 30-nucleotide insertion variant is expressed at a low level. Moreover, Ca2+ release in response to the RyR1 agonist 4-chloro-m-cresol and to thapsigargin showed that the c.7035C>A (p.S2345R) variant causes depletion of S/ER Ca2+ stores and that the compound heterozygosity for variant c.9293G>T (p.S3098I) and the 30-nucleotide insertion increases RyR1-dependent Ca2+ release without affecting ER Ca2+ stores. In conclusion, we detected and functionally characterized disease-causing variants of the RyR1 channel in patient-specific lymphoblastoid cells. This paper is dedicated to the memory and contribution of Luigi Del Vecchio.