María Concepción Gil-Rodríguez

Mutations and Variants in the Cohesion Factor Genes NIPBL, SMC1A, and SMC3 in a Cohort of 30 Unrelated Patients With Cornelia de Lange Syndrome

Cornelia de Lange syndrome (CdLS) manifests facial dysmorphic features, growth and cognitive impairment, and limb malformations. Mutations in three genes (NIPBL, SMC1A, and SMC3) of the cohesin complex and its regulators have been found in affected patients. Here, we present clinical and molecular characterization of 30 unrelated patients with CdLS. Eleven patients had mutations in NIPBL (37%) and three patients had mutations in SMC1A (10%), giving an overall rate of mutations of 47%. Several patients shared the same mutation in NIPBL (p.R827GfsX2) but had variable phenotypes, indicating the influence of modifiers in CdLS. Patients with NIPBL mutations had a more severe phenotype than those with mutations in SMC1A or those without identified mutations. However, a high incidence of palate defects was noted in patients with SMC1A mutations. In addition, we observed a similar phenotype in both male and female patients with SMC1A mutations. Finally, we report the first patient with an SMC1A mutation and the Sandifer complex.

Loss-of-function HDAC8 mutations cause a phenotypic spectrum of Cornelia de Lange syndrome-like features, ocular hypertelorism, large fontanelle and X-linked inheritance

Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder with distinct facies, growth failure, intellectual disability, distal limb anomalies, gastrointestinal and neurological disease. Mutations in NIPBL, encoding a cohesin regulatory protein, account for >80% of cases with typical facies. Mutations in the core cohesin complex proteins, encoded by the SMC1A, SMC3 and RAD21 genes, together account for ∼5% of subjects, often with atypical CdLS features. Recently, we identified mutations in the X-linked gene HDAC8 as the cause of a small number of CdLS cases. Here, we report a cohort of 38 individuals with an emerging spectrum of features caused by HDAC8 mutations. For several individuals, the diagnosis of CdLS was not considered prior to genomic testing. Most mutations identified are missense and de novo. Many cases are heterozygous females, each with marked skewing of X-inactivation in peripheral blood DNA. We also identified eight hemizygous males who are more severely affected. The craniofacial appearance caused by HDAC8 mutations overlaps that of typical CdLS but often displays delayed anterior fontanelle closure, ocular hypertelorism, hooding of the eyelids, a broader nose and dental anomalies, which may be useful discriminating features. HDAC8 encodes the lysine deacetylase for the cohesin subunit SMC3 and analysis of the functional consequences of the missense mutations indicates that all cause a loss of enzymatic function. These data demonstrate that loss-of-function mutations in HDAC8 cause a range of overlapping human developmental phenotypes, including a phenotypically distinct subgroup of CdLS.

De novo heterozygous mutations in SMC3 cause a range of Cornelia de Lange syndrome-overlapping phenotypes.

Cornelia de Lange syndrome (CdLS) is characterized by facial dysmorphism, growth failure, intellectual disability, limb malformations, and multiple organ involvement. Mutations in five genes, encoding subunits of the cohesin complex (SMC1A, SMC3, RAD21) and its regulators (NIPBL, HDAC8), account for at least 70% of patients with CdLS or CdLS‐like phenotypes. To date, only the clinical features from a single CdLS patient with SMC3 mutation has been published. Here, we report the efforts of an international research and clinical collaboration to provide clinical comparison of 16 patients with CdLS‐like features caused by mutations in SMC3. Modeling of the mutation effects on protein structure suggests a dominant‐negative effect on the multimeric cohesin complex. When compared with typical CdLS, many SMC3‐associated phenotypes are also characterized by postnatal microcephaly but with a less distinctive craniofacial appearance, a milder prenatal growth retardation that worsens in childhood, few congenital heart defects, and an absence of limb deficiencies. While most mutations are unique, two unrelated affected individuals shared the same mutation but presented with different phenotypes. This work confirms that de novo SMC3 mutations account for ∼1%–2% of CdLS‐like phenotypes.

Isolated NIBPL missense mutations that cause Cornelia de Lange syndrome alter MAU2 interaction

Cornelia de Lange syndrome (CdLS; or Brachmann-de Lange syndrome) is a dominantly inherited congenital malformation disorder with features that include characteristic facies, cognitive delays, growth retardation and limb anomalies. Mutations in nearly 60% of CdLS patients have been identified in NIPBL, which encodes a regulator of the sister chromatid cohesion complex. NIPBL, also known as delangin, is a homolog of yeast and amphibian Scc2 and C. elegans PQN-85. Although the exact mechanism of NIPBL function in sister chromatid cohesion is unclear, in vivo yeast and C. elegans experiments and in vitro vertebrate cell experiments have demonstrated that NIPBL/Scc2 functionally interacts with the MAU2/Scc4 protein to initiate loading of cohesin onto chromatin. To test the significance of this model in the clinical setting of CdLS, we fine-mapped the NIBPL–MAU2 interaction domain and tested the functional significance of missense mutations and variants in NIPBL and MAU2 identified in these minimal domains in a cohort of patients with CdLS. We demonstrate that specific novel mutations at the N-terminus of the MAU2-interacting domain of NIBPL result in markedly reduced MAU2 binding, although we appreciate no consistent clinical difference in the small group of patients with these mutations. These data suggest that factors in addition to MAU2 are essential in determining the clinical features and severity of CdLS.

Characterization of splice variants of the genes encoding human mitochondrial HMG-CoA lyase and HMG-CoA synthase, the main enzymes of the ketogenesis pathway

The genes HMGCS2 and HMGCL encode the two main enzymes for ketone-body synthesis, mitochondrial HMG-CoA synthase and HMG-CoA lyase. Here, we identify and describe possible splice variants of these genes in human tissues. We detected an alternative transcript of HMGCS2 carrying a deletion of exon 4, and two alternative transcripts of HMGCL with deletions of exons 5 and 6, and exons 5, 6 and 7, respectively. All splice variants maintained the reading frame. However, Western blot studies and overexpression measurements in eukaryotic or prokaryotic cell models did not reveal HL or mHS protein variants. Both genes showed a similar distribution of the inactive variants in different tissues. Surprisingly, the highest percentages were found in tissues where almost no ketone bodies are synthesized: heart, skeletal muscle and brain. Our results suggest that alternative splicing might coordinately block the two main enzymes of ketogenesis in specific human tissues.

Somatic mosaicism in a Cornelia de Lange syndrome patient with NIPBL mutation identified by different next generation sequencing approaches

To the Editor : Cornelia de Lange Syndrome (CdLS) is an autosomal dominant (NIPBL, SMC3 and RAD21 ) or Xlinked (SMC1A and HDAC8 ) congenital disorder, characterized by distinctive craniofacial appearance, growth retardation, intellectual disability and limb malformations (1). Currently, mutations in about 70% of the patients studied have been identified (1). However, recent studies have found limitations in mutation detection efficiency by classical sequencing approaches using DNA derived from peripheral blood samples (2). Thus, Huisman et al. identified NIPBL mutations in buccal epithelial cells of patients with classic CdLS phenotype who were previously negative by classical sequencing approaches using DNA of blood leukocytes (3). This finding supported the hypothesis that somatic mosaicism might contribute to the wide clinical expressivity and to explain the non-detectability of mutations in CdLS patients by classical molecular testing (4, 5). Here we report on a patient with classic CdLS (Fig. 1a i–iii) in whom previous molecular analyses of all known CdLS genes by Sanger sequencing did not identify any mutation. Interestingly, subsequent exome and panel sequencing approaches were able to disclose a somatic mosaicism for a mutation in NIPBL. In addition, pyrosequencing assays were performed to precisely quantify the percentage of the mutated allele in DNA samples from multiple tissues. Sequencing analyses of the five genes associated with CdLS were performed on DNA from blood leukocytes, but Sanger sequencing failed to identify pathological variants in these genes. Thereafter exome sequencing was performed in the affected individual. This technique found a de novo heterozygous missense mutation in exon 39 of the NIPBL gene [c.6647A > C; p.(Tyr2216Ser)]. The mutated allele was present in 5 of 21 (24%) reads in exome sequencing and a weak signal was visible in the chromatogram of the second test of direct sequencing (Fig. 1b). Together, these data prompted us to suspect mosaicism for this NIPBL mutation. Additionally genomic DNA was sequenced using an Ion AmpliseqTM (Life Technologies, Grand Island, NY) custom designed gene panel to amplify the coding exons of 16 genes including the known CdLS genes. After data analysis and database filtering using the Torrent Variant Caller and seqnext software in parallel, only the c.6647A > C mutation in exon 39 of the NIPBL gene was identified (Fig. 1c). Subsequently, pyrosequencing was used for detailed quantification comparing the wild-type (wt) and mutant allele in genomic DNA samples from different tissues. The results revealed the tissue-specific levels of heterozygosity (Fig. 1b). It has been suggested that a significant number of CdLS individuals with negative standard molecular analyses may have somatic mosaicism (3). Among the viable explanations are, the limited sensitivity of Sanger sequencing for detection of somatic mosaicism (lowest level of identifiable mosaicism is between 10% and 20%) and more recently Huisman et al. has proposed the loss of mutations in leukocytes due to reversion and leukocyte specific selection against mutant cells (2, 3). Interestingly, somatic mosaicism in other CdLS causative genes has not been reported so far. Although no elucidation can be stated here, this finding is probably based on the limited diversity of tissues investigated but possibly could also be explained by a NIPBL specific molecular mechanism that is currently unknown. Currently the exome sequencing as well as gene panel enriched sequencing, has been described as very sensitive tools for mosaic mutation detection. Whole exome sequencing has emerged as a novel technique for the identification of mutations even for diagnostic purposes. Although the last years show a dramatic decrease in cost of the exome sequencing, subsequent bioinformatics analysis are limiting for most of the diagnostic laboratories (2). Molecular analysis by gene panel specific sequencing represents a less cost intensive alternative for gene specific sequencing with a very high coverage, and will allow the identification of somatic mosaicism as shown here. On the basis of the data available and the findings presented here, we agree with the recommendation made by Huisman et al., in any patient with clinical

Cornelia de Lange syndrome with NIPBL mutation and mosaic Turner syndrome in the same individual

BackgroundCornelia de Lange syndrome (CdLS) is a dominantly inherited disorder characterized by facial dysmorphism, growth and cognitive impairment, limb malformations and multiple organ involvement. Mutations in NIPBL gene account for about 60% of patients with CdLS. This gene encodes a key regulator of the Cohesin complex, which controls sister chromatid segregation during both mitosis and meiosis. Turner syndrome (TS) results from the partial or complete absence of one of the X chromosomes, usually associated with congenital lymphedema, short stature, and gonadal dysgenesis.Case presentationHere we report a four-year-old female with CdLS due to a frameshift mutation in the NIPBL gene (c.1445_1448delGAGA), who also had a tissue-specific mosaic 45,X/46,XX karyotype. The patient showed a severe form of CdLS with craniofacial dysmorphism, pre- and post-natal growth delay, cardiovascular abnormalities, hirsutism and severe psychomotor retardation with behavioural problems. She also presented with minor clinical features consistent with TS, including peripheral lymphedema and webbed neck. The NIPBL mutation was present in the two tissues analysed from different embryonic origins (peripheral blood lymphocytes and oral mucosa epithelial cells). However, the percentage of cells with monosomy X was low and variable in tissues. These findings indicate that, ontogenically, the NIPBL mutation may have appeared before the mosaic monosomy X.ConclusionsThe coexistence in several patients of these two rare disorders raises the issue of whether there is indeed a cause-effect association. The detailed clinical descriptions indicate predominant CdLS phenotype, although additional TS manifestations may appear in adolescence.

Functional characterization of NIPBL physiological splice variants and eight splicing mutations in patients with cornelia de lange syndrome

Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by distinctive craniofacial features, growth retardation, cognitive impairment, limb defects, hirsutism, and multisystem involvement. Mutations in five genes encoding structural components (SMC1A, SMC3, RAD21) or functionally associated factors (NIPBL, HDAC8) of the cohesin complex have been found in patients with CdLS. In about 60% of the patients, mutations in NIPBL could be identified. Interestingly, 17% of them are predicted to change normal splicing, however, detailed molecular investigations are often missing. Here, we report the first systematic study of the physiological splicing of the NIPBL gene, that would reveal the identification of four new splicing isoforms ΔE10, ΔE12, ΔE33,34, and B’. Furthermore, we have investigated nine mutations affecting splice-sites in the NIPBL gene identified in twelve CdLS patients. All mutations have been examined on the DNA and RNA level, as well as by in silico analyses. Although patients with mutations affecting NIPBL splicing show a broad clinical variability, the more severe phenotypes seem to be associated with aberrant transcripts resulting in a shift of the reading frame.

New case of mitochondrial HMG-CoA synthase deficiency: Functional analysis of eight mutations

Mitochondrial HMG-CoA synthase deficiency is a rare inherited metabolic disorder that affects ketone-body synthesis. Acute episodes include vomiting, lethargy, hepatomegaly, hypoglycaemia, dicarboxylic aciduria, and in severe cases, coma. This deficiency may have been under-diagnosed owing to the absence of specific clinical and biochemical markers, limitations in liver biopsy and the lack of an effective method of expression and enzyme assay for verifying the mutations found. To date, eight patients have been reported with nine allelic variants of the HMGCS2 gene. We present a new method of enzyme expression and a modification of the activity assay that allows, for first time, the functional study of missense mutations found in patients with this deficiency. Four of the missense mutations (p.V54M, p.R188H, p.G212R and p.G388R) did not produce proteins that could have been detected in soluble form by western blot; three produced a total loss of activity (p.Y167C, p.M307T and p.R500H) and one, variant p.F174L, gave an enzyme with a catalytic efficiency of 11.5%. This indicates that the deficiency may occur with partial loss of activity of enzyme. In addition, we describe a new patient with this deficiency, in which we detected the missense allelic variant, c.1162G>A (p.G388R) and the nonsense variant c.1270C>T (p.R424X).

Could a patient with SMC1A duplication be classified as a human cohesinopathy

The disorders caused by mutations in genes encoding subunits and accessory proteins of cohesin complex are collectively termed as cohesinopathies. The best known cohesinopathy is Cornelia de Lange Syndrome (CdLS), which is a multisystem developmental disorder characterized by facial dysmorphism, limb malformations, growth and cognitive impairment. Mutations in five genes, encoding subunits of the cohesin complex (SMC1A, SMC3, RAD21) and its regulators (NIPBL, HDAC8), are responsible for ∼70% of CdLS cases. We describe a 16‐year‐old boy with facial dysmorphism, growth retardation, intellectual disability, hirsutism and small hands, who has a small Supernumerary Marker Chromosome (sSMC) present in mosaic form. sSMC is composed of two duplicated segments encompassing 17 genes including SMC1A gene, at the regions Xp11.22 and Xp11.21q11.1. Clinical comparison between our patient with a previously reported individual with a SMC1A duplication and four male carriers of similar sSMC reported in databases, suggest that they all share clinical features related to cohesinopathies. Although our patient does not have the classical CdLS craniofacial phenotype, he has pre and postnatal growth retardation, intellectual disability and mild musculoskeletal anomalies, features commonly seen in patients with cohesinopathies.