Carolina Baquero-Montoya

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.

Clinical utility gene card for: Cornelia de Lange syndrome

1.5 Mutational spectrum CdLS is a congenital autosomal dominant (NIPBL, SMC3 and RAD21) or X-linked dominant (SMC1A and HDAC8) disorder characterized by facial dysmorphism, preand post-natal growth retardation, developmental, intellectual disability, and multiorgan involvement.1,2 Currently, it is estimated that ~ 80% of patients with CdLS have an identifiable mutation in the NIPBL gene, including the 23% of cases with somatic mosaicism.3,4 More than 300 mutations have been found spreaded throughout the gene, although exon 10 appears to be a hot spot.5 Different types of point mutations have been reported, including (in order of decreasing prevalence) frameshift (32%), missense (26%), nonsense (18%), splice site (17%) and in-frame deletion or insertion (7%). Moreover, 33 larger deletions and one balanced translocation have been reported as well. A much smaller percentage of patients (~4–6%) have mutations in the cohesin complex gene SMC1A,6–8 in which 34 mutations have been identified so far,9 including missense mutations (82%) or in-frame deletions (18%). No frameshift or nonsense mutations have been reported in this gene, possibly because they are lethal or lead to a different yet unrecognisable phenotype.5 To date, only six patients with mutation in SMC3 have been identified.8,10 Mutations in RAD21 have been reported in eight patients with a CdLS phenotype: four whole-gene deletions, two missense, one inframe exonic deletion and one frameshift mutation.11,12 Mutations in the HDAC8 gene account for ~ 4% of mutations in individuals, including a phenotypically distinct subgroup of CdLS.13,14 Reported mutations include missense (the most frequent type), chromosomal microdeletions or microduplications, nonsense and splice site. HDAC8 mutations have also been found in seven males from the same family affected by intellectual disability, short stature, truncal obesity, craniofacial dysmorphic features, gynaecomastia and hypogonadism, overlapping the X-linked intellectual disability syndromes Wilson–Turner syndrome and Börjeson–Forssman–Lehmann syndrome.15 For the standard reference sequence in relation to the variants reported, NCBI Reference Sequences: NM_133433.3 (NIPBL gene), NM_006306.3 (SMC1A), NM_005445.3 (SMC3), NM_006265.2 (RAD21) and NM_018486.2 (HDAC8) should be applied. NIPBL gene variants can be found in the database: Leiden Open Variation Database (LOVD) (http://grenada.lumc.nl/LOVD2/CDLS/ home.php? select_db=NIPBL). Although most of the identified mutations in the CdLS-related genes are considered de novo, familial cases have been reported in patients with mutations in NIPBL,2,16–19 SMC1A,7,9 RAD2112 and HDAC8.14 Although the vast majority of patients with the classic/severe CdLS phenotype carry an identifiable mutation in NIPBL, in some of them

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

Mutations in chromatin regulators functionally link Cornelia de Lange syndrome and clinically overlapping phenotypes

The coordinated tissue-specific regulation of gene expression is essential for the proper development of all organisms. Mutations in multiple transcriptional regulators cause a group of neurodevelopmental disorders termed “transcriptomopathies” that share core phenotypical features including growth retardation, developmental delay, intellectual disability and facial dysmorphism. Cornelia de Lange syndrome (CdLS) belongs to this class of disorders and is caused by mutations in different subunits or regulators of the cohesin complex. Herein, we report on the clinical and molecular characterization of seven patients with features overlapping with CdLS who were found to carry mutations in chromatin regulators previously associated to other neurodevelopmental disorders that are frequently considered in the differential diagnosis of CdLS. The identified mutations affect the methyltransferase-encoding genes KMT2A and SETD5 and different subunits of the SWI/SNF chromatin-remodeling complex. Complementary to this, a patient with Coffin–Siris syndrome was found to carry a missense substitution in NIPBL. Our findings indicate that mutations in a variety of chromatin-associated factors result in overlapping clinical phenotypes, underscoring the genetic heterogeneity that should be considered when assessing the clinical and molecular diagnosis of neurodevelopmental syndromes. It is clear that emerging molecular mechanisms of chromatin dysregulation are central to understanding the pathogenesis of these clinically overlapping genetic disorders.

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.

Special cases in Cornelia de Lange syndrome: The Spanish experience

Cornelia de Lange Syndrome (CdLS) is an autosomal dominant (NIPBL, SMC3, and RAD21) or X‐linked (SMC1A and HDAC8) disorder, characterized by distinctive craniofacial appearance, growth retardation, intellectual disability, and limb anomalies. In 2005, the Spanish CdLS Reference Center was started and now we have more than 270 cases in our database. In this special issue, we describe some of the unique or atypical patients studied by our group, whose clinical features have contributed to the expansion of the CdLS classical phenotype, helping clinicians to diagnose it. We include the case of a male with unilateral tibial hypoplasia and peroneal agenesis who had a mutation in NIPBL; we also describe one patient with a mutation in NIPBL and somatic mosaicism identified by new generation sequencing techniques; we also include one patient with CdLS and Turner syndrome; and last, an interesting patient with a duplication of the SMC1A gene. Finally, we make a short review of the splicing mutations we have found in NIPBL regarding the new knowledge on the physiological variants of the gene.

Identification and Functional Characterization of Two Intronic NIPBL Mutations in Two Patients with Cornelia de Lange Syndrome

Cornelia de Lange syndrome (CdLS) is a rare genetically heterogeneous disorder with a high phenotypic variability including mental retardation, developmental delay, and limb malformations. The genetic causes in about 30% of patients with CdLS are still unknown. We report on the functional characterization of two intronic NIPBL mutations in two patients with CdLS that do not affect a conserved splice-donor or acceptor site. Interestingly, mRNA analyses showed aberrantly spliced transcripts missing exon 28 or 37, suggesting the loss of the branch site by the c.5329-15A>G transition and a disruption of the polypyrimidine by the c.6344del(-13)_(-8) deletion. While the loss of exon 28 retains the reading frame of the NIBPL transcript resulting in a shortened protein, the loss of exon 37 shifts the reading frame with the consequence of a premature stop of translation. Subsequent quantitative PCR analysis demonstrated a 30% decrease of the total NIPBL mRNA levels associated with the frameshift transcript. Consistent with our results, this patient shows a more severe phenotype compared to the patient with the aberrant transcript that retains its reading frame. Thus, intronic variants identified by sequencing analysis in CdLS diagnostics should carefully be examined before excluding them as nonrelevant to disease.

Severe ipsilateral musculoskeletal involvement in a Cornelia de Lange patient with a novel NIPBL mutation.

Cornelia de Lange Syndrome (CdLS) is a congenital autosomal dominant (NIPBL, SMC3 and RAD21) or X-linked (SMC1A and HDAC8) disorder characterized by facial dysmorphism, pre and postnatal growth retardation, developmental delay and/or intellectual disability, and multiorgan involvement. Musculoskeletal malformations are usually bilateral and affect mainly the upper limbs; the range goes from brachyclinodactyly to severe reduction defects. Instead lower extremities are usually less and mildly involved. Here, we report on a 3-year-old Senegalese boy with typical craniofacial CdLS features, pre and postnatal growth retardation, atrial septal defect, developmental delay and right ipsilateral limb malformations, consistent with oligodactyly of the 3rd and 4th fingers, tibial agenesis and fibula hypoplasia. Exome sequencing and Sanger sequencing showed a novel missense mutation in NIPBL gene (c.6647A>G; p.(Tyr2216Cys)), which affects a conserved residue located within NIPBL HEAT repeat elements. Pyrosequencing analysis of NIPBL gene, disclosed similar levels of wild-type and mutated alleles in DNA and RNA samples from all tissues analyzed (oral mucosa epithelial cells, peripheral blood leukocytes and fibroblasts). These findings indicated the absence of somatic mosaicism, despite of the segmental asymmetry of the limbs, and confirmed biallelic expression for NIPBL transcripts, respectively. Additionally, conditions like Split-hand/foot malformation with long-bone deficiency secondary to duplication of BHLHA9 gene have been ruled out by the array-CGH and MLPA analysis. To our knowledge, this is the first CdLS patient described with major ipsilateral malformations of both the upper and lower extremities, that even though this finding could be due to a random event, expands the spectrum of limb reduction defects in CdLS.

mRNA Quantification of NIPBL Isoforms A and B in Adult and Fetal Human Tissues, and a Potentially Pathological Variant Affecting Only Isoform A in Two Patients with Cornelia de Lange Syndrome

Cornelia de Lange syndrome (CdLS) is a congenital developmental disorder characterized by craniofacial dysmorphia, growth retardation, limb malformations, and intellectual disability. Approximately 60% of patients with CdLS carry a recognizable pathological variant in the NIPBL gene, of which two isoforms, A and B, have been identified, and which only differ in the C-terminal segment. In this work, we describe the distribution pattern of the isoforms A and B mRNAs in tissues of adult and fetal origin, by qPCR (quantitative polymerase chain reaction). Our results show a higher gene expression of the isoform A, even though both seem to have the same tissue distribution. Interestingly, the expression in fetal tissues is higher than that of adults, especially in brain and skeletal muscle. Curiously, the study of fibroblasts of two siblings with a mild CdLS phenotype and a pathological variant specific of the isoform A of NIPBL (c.8387A > G; P.Tyr2796Cys), showed a similar reduction in both isoforms, and a normal sensitivity to DNA damage. Overall, these results suggest that the position of the pathological variant at the 3´ end of the NIPBL gene affecting only isoform A, is likely to be the cause of the atypical mild phenotype of the two brothers.