Heterozygous germline mutations in the proto-oncogene HRAS cause Costello syndrome (CS), an intellectual disability condition with severe failure-to-thrive, cardiac abnormalities, predisposition to tumors and neurologic abnormalities. More than 80% of patients share the HRAS mutation c.34G>A (p.Gly12Ser) associated with the typical, relatively homogeneous phenotype. Rarer mutations occurred in individuals with an attenuated phenotype and less characteristic facial features. Most pathogenic HRAS alterations affect hydrolytic HRAS activity resulting in constitutive activation. ‘Gain-of-function’ and ‘hyperactivation’ concerning downstream pathways are widely used to explain the molecular basis and dysregulation of the RAS-MAPK pathway. This is the biologic mechanism shared amongst rasopathies. Panel testing for rasopathies identified a novel HRAS mutation (c.179G>A; p.Gly60Asp) in three individuals with an attenuated CS phenotype. De novo paternal origin was documented in two, transmission from a heterozygous mother occurred in the third. Individuals showed subtle facial features; curly hair and relative macrocephaly were seen in three; atrial tachycardia and learning differences in two, and pulmonic valve dysplasia and mildly thickened left ventricle in one. None had severe failure-to-thrive, intellectual disability or cancer. Functional studies revealed strongly increased HRASGly60Asp binding to RAF1, but not to other signaling effectors. Hyperactivation of the MAPK downstream signaling pathways was absent. Our results and literature data indicate dominant negative consequences of HRAS glycine 60 substitutions on RAS-dependent signaling. We conclude that hyperactivation of RAS downstream signaling does not entirely explain the molecular basis of CS and support the new idea of disrupted HRAS reactivity as a critical molecular dysfunction.

Megacystis‐microcolon‐intestinal hypoperistalsis syndrome (MMIHS), a rare condition that affects smooth muscle cells, is caused by biallelic null alleles in MYH11. We report on a girl with MMIHS in addition to growth hormone deficiency, central hypothyroidism and a tonically dilated pupil with accommodation deficit. Sanger sequencing and arrayCGH uncovered the novel heterozygous missense variant c.379C>T in MYH11 and a heterozygous 1.3 Mb deletion in 16q13.11 encompassing MYH11, respectively. Her mother carries the deletion, whereas her father is heterozygous for the c.379C>T p.(Pro127Ser) change. Proline 127 is crucial for the formation of the Adenosine triphosphate binding pocket of the MYH11 motor domain and molecular modeling indicated that p.Pro127Ser alters nucleotide binding properties. Thus, the unusual and complex clinical presentation of the patient results from compound heterozygosity for a 16p13.11 microdeletion including the entire MYH11 gene and a loss‐of‐function missense variant on the remaining MYH11 allele. In conclusion, we recommend genetic testing both for MYH11 sequence alterations and copy number imbalances in individuals with MMIHS and smooth muscle cell‐associated abnormalities in additional organs, that is, multisystemic smooth muscle dysfunction.

Purpose: Heritable factors play an important etiologic role in connective tissue disorders (CTD) with vascular involvement, and a genetic diagnosis is getting increasingly important for gene-tailored, personalized patient management. Methods: We analyzed 32 disease-associated genes by using targeted next-generation sequencing and exome sequencing in a clinically relevant cohort of 199 individuals. We classified and refined sequence variants according to their likelihood for pathogenicity. Results: We identified 1 pathogenic variant (PV; in FBN1 or SMAD3) in 15 patients (7.5%) and ≥1 likely pathogenic variant (LPV; in COL3A1, FBN1, FBN2, LOX, MYH11, SMAD3, TGFBR1, or TGFBR2) in 19 individuals (9.6%), together resulting in 17.1% diagnostic yield. Thirteen PV/LPV were novel. Of PV/LPV-negative patients 47 (23.6%) showed ≥1 variant of uncertain significance (VUS). Twenty-five patients had concomitant variants. In-depth evaluation of reported/calculated variant classes resulted in reclassification of 19.8% of variants. Conclusion: Variant classification and refinement are essential for shaping mutational spectra of disease genes, thereby improving clinical sensitivity. Obligate stringent multigene analysis is a powerful tool for identifying genetic causes of clinically related CTDs. Nonetheless, the relatively high rate of PV/LPV/VUSnegative patients underscores the existence of yet unknown disease loci and/or oligogenic/polygenic inheritance.