Petrillo Nadia, Marcella Simone, Sirica Roberto, Ianniello Monica, Ruggiero Raffaella, Mori Alessio, Castiello Rosa, Ramiro Cristina, D'Angelo Rossana, Pennacchio Giuliano, Barletta Ermanno, Passaro Roberto, Fico Antonio, Savarese Giovanni
AMES, Centro Polidiagnostico Strumentale s.r.l., Via Padre Carmine Fico 24, 80013 Casalnuovo Di Napoli, Italy.
CEINGE Biotecnologie Avanzate Franco Salvatore S.c.a.r.l., 80145 Naples, Italy.
Genes (Basel). 2025 Mar 5;16(3):311. doi: 10.3390/genes16030311.
BACKGROUND/OBJECTIVES: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by mutations in the gene associated with 21-hydroxylase deficiency and increased levels of adrenal androgens. Affected females are at risk of ambiguous genitalia, while affected males show sexual precocity. Here, we present a case of a newborn female patient, characterized by ambiguous genitalia and previously identified as low risk for common aneuploidies by non-invasive prenatal testing (NIPT).
We performed a NIPT, which showed a 46, XX genotype, confirmed by karyotype on the newborn's DNA extracted lymphocytes. For clinical suspicion of CAH, we performed reverse dot blot and Multiple Ligation-dependent Probe Amplification (MLPA) of the gene on the patients and her parents' DNA. Then, we performed on mother's plasma NGS analysis with an in-house developed panel of genes for monogenic diseases, including the gene.
Reverse dot blot and MLPA detected the presence of the c.290-13A/C>G (I2 splice) mutation in heterozygosity in the parents and in homozygosity in the child, respectively. NGS detected the c.290-13A/C>G (I2splice) mutation in cell-free fetal DNA (cfDNA) in mother's plasma with a variant allele frequency (VAF) of 67% with a fetal fraction (FF) of 5%. This latter suggests the presence of the variant both in the mother and in newborn cfDNA.
The study reinforces the hypothesis that cfDNA can be used to identify point mutations, small insertions and/or deletions for the diagnosis of monogenic diseases, reducing the number of invasive tests and the risk of early miscarriages. Early detection of mutations in genes causing sexual development disorders could make it possible to start therapy in the womb.
背景/目的：先天性肾上腺皮质增生症（CAH）是一种常染色体隐性疾病，由与21 - 羟化酶缺乏相关的基因突变以及肾上腺雄激素水平升高引起。受影响的女性有生殖器模糊的风险，而受影响的男性表现为性早熟。在此，我们报告一例新生儿女性患者，其特征为生殖器模糊，且通过无创产前检测（NIPT）先前被确定为常见非整倍体低风险。
我们进行了NIPT，结果显示为46, XX基因型，通过对新生儿提取淋巴细胞的DNA进行核型分析得以证实。由于临床怀疑CAH，我们对患者及其父母的DNA进行了该基因的反向点杂交和多重连接依赖探针扩增（MLPA）。然后，我们使用内部开发的单基因疾病基因检测板对母亲的血浆进行了二代测序分析，其中包括该基因。
反向点杂交和MLPA分别检测到父母中该基因杂合存在c.290 - 13A/C>G（I2剪接）突变，而患儿中为纯合突变。二代测序在母亲血浆的游离胎儿DNA（cfDNA）中检测到c.290 - 13A/C>G（I2剪接）突变，变异等位基因频率（VAF）为67%，胎儿游离DNA比例（FF）为5%。这表明母亲和新生儿的cfDNA中均存在该变异。
该研究强化了这样一种假设，即cfDNA可用于识别点突变、小插入和/或缺失以诊断单基因疾病，减少侵入性检测的数量以及早期流产的风险。早期检测导致性发育障碍的基因突变可能使在子宫内开始治疗成为可能。
