Deborah L. Stabley

HRAS mutation analysis in Costello syndrome: Genotype and phenotype correlation

Costello syndrome is a rare condition comprising mental retardation, distinctive facial appearance, cardiovascular abnormalities (typically pulmonic stenosis, hypertrophic cardiomyopathy, and/or atrial tachycardia), tumor predisposition, and skin and musculoskeletal abnormalities. Recently mutations in HRAS were identified in 12 Japanese and Italian patients with clinical information available on 7 of the Japanese patients. To expand the molecular delineation of Costello syndrome, we performed mutation analysis in 34 North American and 6 European (total 40) patients with Costello syndrome, and detected missense mutations in HRAS in 33 (82.5%) patients. All mutations affected either codon 12 or 13 of the protein product, with G12S occurring in 30 (90.9%) patients of the mutation‐positive cases. In two patients, we found a mutation resulting in an alanine substitution in position 12 (G12A), and in one patient, we detected a novel mutation (G13C). Five different HRAS mutations have now been reported in Costello syndrome, however genotype–phenotype correlation remains incomplete.

Further delineation of the phenotype resulting from BRAF or MEK1 germline mutations helps differentiate cardio-facio-cutaneous syndrome from Costello syndrome†‡§

Because Cardio‐facio‐cutaneous (CFC) syndrome has significant phenotypic overlap with Costello syndrome, it may be difficult to establish the diagnosis on a clinical basis. The recent discoveries of germline HRAS mutations in patients with Costello syndrome and mutations in BRAF, MEK1, and MEK2 in CFC syndrome uncovered the biologic mechanism for the shared phenotypic findings based on the close interaction of the affected gene products within the MAP kinase pathway. We evaluated a series of patients who were either clinically diagnosed with Costello syndrome, or in whom the diagnoses of both Costello and CFC syndromes were considered. After excluding mutations in HRAS, we identified eight changes in BRAF and five in MEK1. Five mutations are novel, and all changes occurred de novo among those triads tested. A review of the clinical abnormalities showed important differences between patients with either a BRAF or MEK1 mutation, and those previously reported with an HRAS mutation. Statistical significance was achieved, despite the relatively small number of patients with BRAF and MEK1 mutations reported here, for polyhydramnios, growth hormone deficiency and the presence of more than one papilloma, which were less common in CFC compared to HRAS mutation positive patients. Although both CFC and Costello syndrome are characterized by cardiac abnormalities in about three‐fourths of patients, the pattern of congenital heart defects (CHD), hypertrophic cardiomyopathy (HCM), and tachycardia differs somewhat. CHD, especially pulmonic stenosis associated with a secundum‐type atrial septal defect, are more common in CFC than Costello syndrome (P = 0.02). Atrial tachycardia is less frequent in CFC patients with BRAF or MEK1 mutations, compared to Costello syndrome patients with HRAS mutation (P = 0.04). Chaotic atrial rhythm or multifocal atrial tachycardia was observed only in Costello syndrome. Malignant tumors have been viewed as characteristic for Costello syndrome due to HRAS mutations, however, we report here on a MEK1 mutation in a patient with a malignant tumor, a hepatoblastoma. Although this indicates that the presence of a tumor is not specific for Costello syndrome with HRAS mutation, it is noteworthy that the tumor histology differs from those commonly seen in Costello syndrome. Based on these clinical differences we suggest that patients with BRAF and MEK mutations should be diagnosed with CFC syndrome, and the diagnosis of Costello syndrome be reserved for patients with HRAS mutations.

Somatic mosaicism for an HRAS mutation causes Costello syndrome

De novo heterozygous HRAS point mutations have been reported in more than 81 patients with Costello syndrome (CS), but genotype/phenotype correlation remains incomplete because the majority of patients share a common mutation, G12S, seen in 65/81 (80%). Somatic HRAS mutations have previously been identified in solid tumors, and mutation hot spots related to a gain‐of‐function effect of the gene product are known. The germline mutations causing CS occur at these hot spots and convey a gain‐of‐function effect, thus accounting for the greatly increased cancer risk. Diagnostic testing for HRAS mutations is now available and the identification of a mutation in a patient with consistent clinical findings confirms a diagnosis of CS. It is not clear yet if the absence of an HRAS mutation precludes a diagnosis of CS. Because there is a significant overlap in the clinical findings of Costello, cardio‐facio‐cutaneous, and Noonan syndromes, diagnostic uncertainty remains in patients lacking an HRAS mutation. We report here on a female with findings suggestive of CS in whom mutation analysis performed with standard techniques on white blood cell derived DNA did not show an HRAS mutation. However, analysis of DNA derived from three independently collected buccal swabs showed a sequence change qualitatively consistent with the G12S mutation. Allelic quantitation showed the presence of the mutation in ∼25%–30% of the sampled buccal cells. In this patient, standard technology failed to identify the disease causing mutation on DNA derived from a blood sample, highlighting the potential pitfalls in the interpretation of negative mutation studies. This is the first reported CS patient mosaic for the common HRAS mutation, likely due to a somatic mutation occurring very early in fetal development.

Evolution of placentally expressed cathepsins.

Species and strain variants of a family of placentally expressed cathepsins (PECs) were cloned and sequenced in order to identify evolutionary conserved structural characteristics of this large family of cysteine proteases. Cathepsins M, P, Q, and R, are conserved in mice and rats but homologs of these genes are not found in human or rabbit placenta, showing that this family of proteases are probably restricted to rodents. Species-specific gene duplications have given rise to variants of cathepsin M in mice, and cathepsin Q in rats. Although the PECs have diverged at a greater rate than the other lysosomal cathepsins, residues around the specificity sub-sites of the individual enzymes are conserved. Strain-specific polymorphisms show that the evolutionary rate of divergence of cathepsins M and 3, the most recently duplicated pair of mouse genes, is even higher than the other PECs. In human placenta, critical functions of the PECs are probably performed by broader specificity proteases such as cathepsins B and L.

Costello syndrome associated with novel germline HRAS mutations: An attenuated phenotype?

Costello syndrome is a rare congenital disorder typically characterized by severe failure‐to‐thrive, cardiac abnormalities including tachyarrhythmia and hypertrophic cardiomyopathy, distinctive facial features, a predisposition to papillomata and malignant tumors, neurologic abnormalities, developmental delay, and mental retardation. Its underlying cause is de novo germline mutations in the oncogene HRAS. Almost all Costello syndrome mutations affect one of the glycine residues in position 12 or 13 of the protein product. More than 80% of patients with Costello syndrome share the same underlying mutation, resulting in a G12S amino acid change. We report on two patients with novel HRAS mutations affecting amino acids 58 (T58I) and 146 (A146V), respectively. Despite facial features that appear less coarse than those typically seen in Costello patients, both patients show many of the physical and developmental problems characteristic for Costello syndrome. These novel HRAS mutations may be less common than the frequently reported G12S change, or patients with these changes may be undiagnosed due to their less coarse facial features. In addition to the findings previously known to occur in Costello syndrome, one of our patients had hypertrophic pyloric stenosis. This led us to review the medical histories on a cohort of proven HRAS mutation positive Costello syndrome patients, and we found a statistically significantly (P < 0.001) increased frequency of pyloric stenosis in Costello syndrome (5/58) compared to the general population frequency of 2–3/1,000. Thus we add hypertrophic pyloric stenosis to the abnormalities seen with increased frequency in Costello syndrome.

Univariate and bivariate variance component linkage analysis of a whole-genome scan for loci contributing to bone mineral density

Osteoporosis is a common condition characterized by reduced skeletal strength and increased susceptibility to fracture. The single major risk factor for osteoporosis is low bone mineral density (BMD) and strong evidence exists that genetic factors are in part responsible for an individuals BMD. A cohort of 40 multiplex Caucasian families selected through a proband with osteoporosis was genotyped for microsatellite markers spaced at an average of 10 cM, and linkage to femoral neck (FN), lumbar spine (LS) and trochanter (TR) BMD was analyzed using univariate and bivariate variance component linkage analysis. Maximum univariate multipoint lod-scores were 2.87 on chromosome 1p36 for FN BMD, 1.89 on 6q27 for TR BMD, and 2.15 on 7p15 for LS BMD. Results of bivariate linkage analysis were highly correlated with those of the univariate analysis, although generally less significant, suggesting the possibility that some of these susceptibility loci may exert pleiotropic effects on multiple skeletal sites.

Diamond–Blackfan anemia with mandibulofacial dystostosis is heterogeneous, including the novel DBA genes TSR2 and RPS28

Patients with physical findings suggestive of Treacher Collins syndrome (TCS) or mandibulofacial dysostosis (MFD) and macrocytic anemia diagnostic of Diamond–Blackfan anemia (DBA) have been reported. Disease‐causing genes have been identified for TCS and other MFDs. Mutations in several ribosomal protein genes and the transcription factor GATA1 result in DBA. However, no disease‐causing mutation had been identified in the reported patients with the combination of TCS/MFD and DBA phenotype, and we hypothesized that pathogenic mutations in a single gene could be identified using whole exome analysis. We studied probands from six unrelated families. Combining exome analysis and Sanger sequencing, we identified likely pathogenic mutations in 5/6 families. Two mutations in unrelated families were seen in RPS26, the known DBA10 gene. One variant was predicted to affect mRNA splicing, and the other to lead to protein truncation. In another family a likely pathogenic X‐linked mutation affecting a highly conserved residue was found in TSR2, which encodes a direct binding partner of RPS26. De novo mutations affecting the RPS28 start codon were found in two unrelated probands, identifying RPS28 as a novel disease gene. We conclude that the phenotype combining features of TCS with DBA is genetically heterogeneous. Each of the pathogenic variants identified is predicted to impede ribosome biogenesis, which in turn could result in altered cell growth and proliferation, causing abnormal embryologic development, defective erythropoiesis and reduced growth. The phenotype combining TCS/MFD and DBA is highly variable, overlaps with DBA and lies within the phenotypic spectrum of ribosomopathies.

Male-to-male transmission of Costello syndrome: G12S HRAS germline mutation inherited from a father with somatic mosaicism†‡

Costello syndrome is a rare congenital anomaly syndrome associated with mental retardation and predisposition to benign and malignant tumors, caused by heterozygous missense mutations in the HRAS oncogene. Previously, all molecularly analyzed mutations appeared de novo, and most arose in the paternal germline. A single patient with somatic mosaicism for a Costello syndrome causing HRAS mutation has been reported. Here we describe the first documented transmission of an HRAS mutation from a parent with somatic mosaicism to a child with typical Costello syndrome. Prior to the identification of the underlying gene mutation in Costello syndrome, this family had been identified clinically. The proband was subsequently found to carry a G12S HRAS germline mutation. Testing of the parents for parental origin identified his father as mosaic for the same HRAS mutation. The mother was found not to carry an HRAS mutation. The causative familial mutation is identified as a c.34G > A, which is the most common mutation in the HRAS gene in patients with Costello syndrome. The father carries the mutation in 7–8% of his alleles. This is the second case of mosaicism observed in Costello syndrome and the first direct molecular evidence of father‐to‐son transmission of the disease‐causing mutation. Our observation underlines the importance of parental evaluation, and may have implications for genetic counseling and clinical practice.

A PLP splicing abnormality is associated with an unusual presentation of PMD.

We report that a deletion of 19 base pairs (bp) in intron 3 of the proteolipid protein (PLP/DM20) gene causes a neurological disease characterized by mild developmental delay, followed by progressive decline of acquired motor and cognitive milestones. The clinical features are associated with mild delay in myelination demonstrated by magnetic resonance imaging studies and with ongoing demyelination and axonal loss demonstrated by magnetic resonance spectroscopy. We demonstrate that the purine‐rich 19bp element regulates PLP‐specific splice site selection in transient transfections of chimeric constructs into cultured oligodendrocytes. Runs of 4 and 5 Gs centered in the 19bp element are critical for efficient PLP‐specific splicing. The intronic element is sequence specific in oligodendrocytes and is not a repressor of PLP‐specific splicing in nonglial cells. These data support the conclusion that deletion of the 19bp purine‐rich region in PLP intron 3 causes a reduction in PLP message and protein, which affects myelin stability and axonal integrity.

A new major histocompatibility complex class I b gene expressed in the mouse blastocyst and placenta

Abstract Because of the role major histocompatibility complex (MHC) class I b molecules may play during mouse embryonic development, we thought it would be interesting to search for additional MHC class I b molecules that might be expressed in preimplantation embryos, and in particular in the trophoblastic lineage. We therefore screened a mouse preimplantation blastocyst cDNA library for MHC class I sequences. This search led to the identification and characterization of a new MHC class I b gene, blastocyst MHC. Sequences identical to the exons and 3′ untranslated region of this gene have been found in many laboratory mouse strains, as well as in the related mouse species Mus spreciligus. The presence of this gene in mouse strains of different MHC class I haplotypes argues that blastocyst MHC is a unique, newly-described gene rather than a new allele of a previously described mouse MHC class I gene. Blastocyst MHC has the structure of an MHC class I b gene, with the six exons characteristic of T-region genes. It is linked to H2-D. The amino acid sequence encoded by this gene maintains all the features of a functional antigen-presentation domain. The blastocyst MHC gene, like the human class I b gene HLA-G, is expressed at the blastocyst stage and in the placenta, and may be the mouse analog for HLA-G.