The L467F-F508del Complex Allele Hampers Pharmacological Rescue of Mutant CFTR by Elexacaftor/Tezacaftor/Ivacaftor in Cystic Fibrosis Patients: The Value of the Ex Vivo Nasal Epithelial Model to Address Non-Responders to CFTR-Modulating Drugs.

Loss-of-function mutations of the CFTR gene cause cystic fibrosis (CF) through a variety of molecular mechanisms involving altered expression, trafficking, and/or activity of the CFTR chloride channel. The most frequent mutation among CF patients, F508del, causes multiple defects that can be, however, overcome by a combination of three pharmacological agents that improve CFTR channel trafficking and gating, namely, elexacaftor, tezacaftor, and ivacaftor. This study was prompted by the evidence of two CF patients, compound heterozygous for F508del and a minimal function variant, who failed to obtain any beneficial effects following treatment with the triple drug combination. Functional studies on nasal epithelia generated in vitro from these patients confirmed the lack of response to pharmacological treatment. Molecular characterization highlighted the presence of an additional amino acid substitution, L467F, in cis with the F508del variant, demonstrating that both patients were carriers of a complex allele. Functional and biochemical assays in heterologous expression systems demonstrated that the double mutant L467F-F508del has a severely reduced activity, with negligible rescue by CFTR modulators. While further studies are needed to investigate the actual prevalence of the L467F-F508del allele, our results suggest that this complex allele should be taken into consideration as plausible cause in CF patients not responding to CFTR modulators.

Variants in the 5'UTR reduce SHOX expression and contribute to SHOX haploinsufficiency.

SHOX haploinsufficiency causes 70-90% of Léri-Weill dyschondrosteosis (LWD) and 2-10% of idiopathic short stature (ISS). Deletions removing the entire gene or enhancers and point mutations in the coding region represent a well-established cause of haploinsufficiency. During diagnostic genetic testing on ISS/LWD patients, in addition to classic SHOX defects, five 5'UTR variants (c.-58G > T, c.-55C > T, c.-51G > A, c.-19G > A, and c.-9del), were detected whose pathogenetic role was unclear and were thus classified as VUS (Variants of Uncertain Significance). The purpose of the present study was to investigate the role of these noncoding variations in SHOX haploinsufficiency. The variants were tested for their ability to interfere with correct gene expression of a regulated reporter gene (luciferase assay). The negative effect on the mRNA splicing predicted in silico for c.-19G > A was assayed in vitro through a minigene splicing assay. The luciferase assay showed that c.-51G > A, c.-19G > A, and c.-9del significantly reduce luciferase activity by 60, 35, and 40% at the homozygous state. Quantification of the luciferase mRNA showed that c.-51G > A and c.-9del might interfere with the correct SHOX expression mainly at the post-transcriptional level. The exon trapping assay demonstrated that c.-19G > A determines the creation of a new branch site causing an aberrant mRNA splicing. In conclusion, this study allowed us to reclassify two of the 5'UTR variants identified during SHOX diagnostic screening as likely pathogenic, one remains as a VUS, and two as likely benign variants. This analysis for the first time expands the spectrum of the genetic causes of SHOX haploinsufficiency to noncoding variations in the 5'UTR.

Skeletal dysplasia with bowing long bones: Proposed flowchart for prenatal diagnosis with case demonstration.

OBJECTIVE: Skeletal dysplasia with bowing long bones is a rare group of multiple characterized congenital anomalies. MATERIALS AND METHODS: We introduce a simple, practical diagnostic flowchart that may be helpful in identifying the appropriate pathway of obstetrical management. RESULTS: Herein, we describe four fetal cases of bent bony dysplasia that focus on ultrasound findings, phenotype, molecular tests, distinctive X-ray features, and chondral growth plate histology. The first case was a typical campomelic dysplasia resulting from a de novo mutation in the SOX9 gene. The second fetus was affected by osteogenesis imperfecta Type II carrying a mutation in the COLA1 gene. The third case was a rare presentation of campomelic dysplasia, Cumming type, in which SOX9 examination was normal. Subsequently, a femoral hypoplasia unusual facies syndrome is also discussed. CONCLUSION: Targeted molecular tests and genetic counseling are required for supplementing ultrasound imaging in order to diagnose the correct skeletal disorders.

Majewski syndrome (short-rib polydactyly syndrome type II): Prenatal diagnosis and histological features of chondral growth plate, liver and kidneys.

The Majewski syndrome or short rib-polydactyly syndrome (SRPS) type II is a lethal skeletal dysplasia characterized by severe IUGR (intrauterine growth restriction) and dysmorphic face, polydactyly, relatively proportionate head size at birth with later progression to microcephaly. A case of second trimester ultrasound diagnosis of SRPS type II is reported with review of the medical record of previous observed cases. Postmortem examination and radiogram confirmed the clinical diagnosis. Histological examination of the femoral epypheseal chondral plate showed an expanded and irregular hypertrophic zone. Moreover, characteristic cortico-medullary cysts of both kidneys and portal fibrosis were also demonstrated; findings consistent with the broad phenotypic spectrum of this rare skeletal disease.

Dysmorphic choroid plexuses and hydrocephalus associated with increased nuchal translucency: early ultrasound markers of de novo thanatophoric dysplasia type II with cloverleaf skull (Kleeblattschaedel).

Prenatal diagnosis of thanatophoric dysplasia (TD) type II presenting in the first trimester with increased nuchal translucency (NT) and cloverleaf skull (Kleeblattschaedel) have been scantly reported in the medical record. Abnormal choroid plexus has been seen in association with fetal anomalies. Here we described a case of increased NT associated with indented choroid plexuses, early onset hydrocephalus and cloverleaf skull in a fetus subsequently diagnosed at early second trimester to carry a de novo mutation encoding for TD type II. The findings of dysmorphic choroid plexus, early onset hydrocephalus and cloverleaf skull at first trimester scan may be early, useful ultrasound markers of TD type II. Molecular analysis to control for possible overlapping syndromes were performed and resulted negative. Postmortem X-ray and 3D-CT scan confirmed the cloverleaf skull, narrow thorax, straight femur with rhizomelic shortening of the limbs and the presence of a communicating hydrocephalus.

p.His165Pro: a novel SOX9 missense mutation of campomelic dysplasia.

Campomelic dysplasia (CD) is a rare skeletal dysplasia caused by mutation in the SOX9 gene located on chromosome 17q24.3-q25.1, which regulates testis and chondrocyte development. Severe bowing of the long bones was seen at second-trimester scan. DNA analysis demonstrated a previously unreported de novo missense mutation in p.His165Pro. Ultrasound-based, molecular biology diagnosis led to early therapeutic termination of pregnancy. Histologic examination of the femoral epyphyseal growth plate confirmed scanty proliferation zone and maturation zone with degenerated chondrocytes.

A de novo balanced translocation t(7;12)(p21.2;p12.3) in a patient with Saethre-Chotzen-like phenotype downregulates TWIST and an osteoclastic protein-tyrosine phosphatase, PTP-oc.

Saethre-Chotzen syndrome (SCS) is an autosomal dominant craniosynostosis syndrome with variable expression. Here we report on a female infant with a de novo balanced translocation 46, XX, t(7;12)(p21.2;p12.3) and presenting at birth brachycephaly, antimongolic palpebral fissures, ocular hypertelorism, broad nose with low nasal bridge and low-set ears. This phenotype is suggestive of a subtle form of SCS, given the absence of limbs anomalies. Cloning of both breakpoints revealed that the translocation does not interrupt the TWIST1 coding region, on 7p21, known to be causative for SCS, but downregulates TWIST1 expression due to a position effect. On chromosome 12, the breakpoint translocates a shorter transcript of PTPRO gene, the osteoclastic protein-tyrosine phosphatase, PTP-oc, near to regulatory region of 7p leading to down-regulation of PTP-oc in the proband's fibroblasts. This is a confirmatory case report providing further evidence for TWIST1 haploinsufficiency in SCS, although a possible role of PTP-oc as genetic factor underlying or at least influencing the development of craniosynostosis could not be a priori excluded.

Craniosynostosis, hydrocephalus, Chiari I malformation and radioulnar synostosis: probably a new syndrome.

We report on clinical and molecular findings of two brothers that both presented with sagittal craniosynostosis, hydrocephalus, Chiari I malformation, blepharophimosis, small low-set ears, hypoplastic philtrum, radioulnar synostosis, kidney malformation, and hypogenitalism. Their father presented mild brachydactyly. Conventional cytogenetic and array CGH screening did not show any chromosomal gains or losses. Furthermore, molecular genetic screening of genes involved in different craniosynostosis syndromes, namely FGFR1, FGFR2, FGFR3, TWIST, RECQL4, and POR genes failed to detect any mutations in genomic DNA. The unique range of clinical manifestations in these two patients and the negative findings of the molecular genetic screening suggest the hypothesis of a previously unrecognized syndrome.