Ina Schanze

Cancer spectrum and frequency among children with Noonan, Costello, and cardio-facio-cutaneous syndromes

Background:Somatic mutations affecting components of the Ras-MAPK pathway are a common feature of cancer, whereas germline Ras pathway mutations cause developmental disorders including Noonan, Costello, and cardio-facio-cutaneous syndromes. These ‘RASopathies’ also represent cancer-prone syndromes, but the quantitative cancer risks remain unknown.Methods:We investigated the occurrence of childhood cancer including benign and malignant tumours of the central nervous system in a group of 735 individuals with germline mutations in Ras signalling pathway genes by matching their information with the German Childhood Cancer Registry.Results:We observed 12 cases of cancer in the entire RASopathy cohort vs 1.12 expected (based on German population-based incidence rates). This corresponds to a 10.5-fold increased risk of all childhood cancers combined (standardised incidence ratio (SIR)=10.5, 95% confidence interval=5.4–18.3). The specific cancers included juvenile myelomonocytic leukaemia=4; brain tumour=3; acute lymphoblastic leukaemia=2; rhabdomyosarcoma=2; and neuroblastoma=1. The childhood cancer SIR in Noonan syndrome patients was 8.1, whereas that for Costello syndrome patients was 42.4.Conclusions:These data comprise the first quantitative evidence documenting that the germline mutations in Ras signalling pathway genes are associated with increased risks of both childhood leukaemia and solid tumours.

STXBP1 encephalopathy A neurodevelopmental disorder including epilepsy

Objective: To give a comprehensive overview of the phenotypic and genetic spectrum of STXBP1 encephalopathy (STXBP1-E) by systematically reviewing newly diagnosed and previously reported patients. Methods: We recruited newly diagnosed patients with STXBP1 mutations through an international network of clinicians and geneticists. Furthermore, we performed a systematic literature search to review the phenotypes of all previously reported patients. Results: We describe the phenotypic features of 147 patients with STXBP1-E including 45 previously unreported patients with 33 novel STXBP1 mutations. All patients have intellectual disability (ID), which is mostly severe to profound (88%). Ninety-five percent of patients have epilepsy. While one-third of patients presented with Ohtahara syndrome (21%) or West syndrome (9.5%), the majority has a nonsyndromic early-onset epilepsy and encephalopathy (53%) with epileptic spasms or tonic seizures as main seizure type. We found no correlation between severity of seizures and severity of ID or between mutation type and seizure characteristics or cognitive outcome. Neurologic comorbidities including autistic features and movement disorders are frequent. We also report 2 previously unreported adult patients with prominent extrapyramidal features. Conclusion: De novo STXBP1 mutations are among the most frequent causes of epilepsy and encephalopathy. Most patients have severe to profound ID with little correlation among seizure onset, seizure severity, and the degree of ID. Accordingly, we hypothesize that seizure severity and ID present 2 independent dimensions of the STXBP1-E phenotype. STXBP1-E may be conceptualized as a complex neurodevelopmental disorder rather than a primary epileptic encephalopathy.

Haploinsufficiency of SOX5, a member of the SOX (SRY-related HMG-box) family of transcription factors is a cause of intellectual disability

Intellectual disability (ID) is a clinically and genetically heterogeneous condition; the cause is unknown in most non-specific and sporadic cases. To establish an etiological basis in those patients represents a difficult challenge. Over the last years it has become apparent that chromosomal rearrangements below the detection level of conventional karyotyping contribute significantly to the cause of ID. We present three patients with non-specific intellectual disability who all have overlapping microdeletions in the chromosomal region 12p12.1. De novo occurrence of the deletion could be proven in the two cases from which parental samples were available. All three identified deletions have different breakpoints and range in size from 120 kb to 4.9 Mb. The smallest deletion helps to narrow down the critical region to a genomic segment (chr12:23,924,800-24,041,698, build 37/hg19) encompassing only one gene, SOX5. SOX5 is a member of the SOX (SRY-related HMG-box) family of transcription factors shown to play roles in chondroblast function, oligodendrocyte differentiation and migration, as well as ensuring proper development of specific neuronal cell types. Because of these biological functions, mutations in SOX5 are predicted to cause complex disease syndromes, as it is the case for other SOX genes, but such mutations have not yet been identified. Our findings indicate that haploinsufficiency of SOX5 is a cause of intellectual disability without any striking physical anomalies.

HIBCH deficiency in a patient with phenotypic characteristics of mitochondrial disorders.

HIBCH (3‐hydroxyisobutyryl‐CoA hydrolase) deficiency (MIM #250620) is a rare autosomal recessive inborn error of metabolism, leading to a block in the catabolic pathway of the amino acid valine and presumably to accumulation of toxic valine metabolites in mitochondria. Only three families with HIBCH deficiency and biallelic HIBCH mutations have been described. We report on a further patient, first child of healthy consanguineous parents, with severe developmental delay, seizures, hyperintensities of the basal ganglia on magnetic resonance imaging (MRI), progressive brain atrophy, optic nerve atrophy, repeatedly elevated blood lactate, and respiratory chain complexes I, I + III and cytochrome c oxidase deficiencies with borderline depletion of mitochondrial DNA in muscle tissue. Laboratory findings in blood and skeletal muscle were inconsistent and did not allow a definite diagnosis, but supported the hypothesis of mitochondrial dysfunction. Homozygosity mapping and whole‐exome sequencing revealed a homozygous one‐base pair insertion in HIBCH. Deficiency of enzyme activity was confirmed in cultured fibroblasts. Although relatively unspecific, the clinical features were similar to those of the previously reported cases. Given the clinical variability and large number of differential diagnoses, the prevalence of HIBCH deficiency is probably underestimated. Next‐generation sequencing approaches are an effective tool for identifying the underlying genetic basis in patients suspected of mitochondrial disorders.

Copy number variants including RAS pathway genes—How much RASopathy is in the phenotype?

The RASopathies comprise a group of clinically overlapping developmental syndromes the common pathogenetic basis of which is dysregulated signal flow through the RAS‐MAPK pathway. Mutations in several components or modifiers of the pathway have been identified in Noonan syndrome and related disorders. Over the past years copy number variants (CNVs) encompassing RAS pathway genes (PTPN11, RAF1, MEK2, or SHOC2) have been reported in children with developmental syndromes. These observations raised speculations that the associated phenotypes represent RASopathies, implying that the increased or reduced expression of the respective RAS pathway component and a consecutive dysregulation of RAS pathway signalling is responsible for the clinical picture. Herein, we present two individuals and three of their relatives harboring duplications of either 3p25.2 including the RAF1 locus or 19p13.3 including the MEK2 locus. Duplication carriers exhibited variable clinical phenotypes including non‐specific facial dysmorphism, short stature, and learning difficulties. A careful review of the literature supported the impression that phenotypes associated with CNVs including RAS pathway genes commonly share non‐specific symptoms with RASopathies, while the characteristic “gestalt” is lacking. Considering the known molecular pathogenesis of RASopathies, it is questionable that a modest increase in the expression of a functionally normal signaling component can mimic the effects of a qualitatively abnormal (hyperactive) mutant protein. We thus argue that current empirical and biological evidence is still insufficient to allow the conclusion that an altered copy number of a RAS pathway component is indeed the mechanism that is critical for the phenotype associated with CNVs including RASopathy genes.

Deletions in the 3′ part of the NFIX gene including a recurrent alu-mediated deletion of exon 6 and 7 account for previously unexplained cases of marshall-smith syndrome

Marshall–Smith syndrome (MSS) is a very rare malformation syndrome characterized by typical craniofacial anomalies, abnormal osseous maturation, developmental delay, failure to thrive, and respiratory difficulties. Mutations in the nuclear factor 1/X gene (NFIX) were recently identified as the cause of MSS. In our study cohort of 17 patients with a clinical diagnosis of MSS, conventional sequencing of NFIX revealed frameshift and splice‐site mutations in 10 individuals. Using multiplex ligation‐dependent probe amplification analysis, we identified a recurrent deletion of NFIX exon 6 and 7 in five individuals. We demonstrate this recurrent deletion is the product of a recombination between AluY elements located in intron 5 and 7. Two other patients had smaller deletions affecting exon 6. These findings show that MSS is a genetically homogeneous Mendelian disorder. RT‐PCR experiments with newly identified NFIX mutations including the recurrent exon 6 and 7 deletion confirmed previous findings indicating that MSS‐associated mutant mRNAs are not cleared by nonsense‐mediated mRNA decay. Predicted MSS‐associated mutant NFIX proteins consistently have a preserved DNA binding and dimerization domain, whereas they grossly vary in their C‐terminal portion. This is in line with the hypothesis that MSS‐associated mutations encode dysfunctional proteins that act in a dominant negative manner.

Loss-of-function variants in HIVEP2 are a cause of intellectual disability

Intellectual disability (ID) affects 2–3% of the population. In the past, many genetic causes of ID remained unidentified due to its vast heterogeneity. Recently, whole exome sequencing (WES) studies have shown that de novo variants underlie a significant portion of sporadic cases of ID. Applying WES to patients with ID or global developmental delay at different centers, we identified three individuals with distinct de novo variants in HIVEP2 (human immunodeficiency virus type I enhancer binding protein), which belongs to a family of zinc-finger-containing transcriptional proteins involved in growth and development. Two of the variants were nonsense changes, and one was a 1 bp deletion resulting in a premature stop codon that was reported previously without clinical detail. In silico prediction programs suggest loss-of-function in the mutated allele leading to haploinsufficiency as a putative mechanism in all three individuals. All three patients presented with moderate-to-severe ID, minimal structural brain anomalies, hypotonia, and mild dysmorphic features. Growth parameters were in the normal range except for borderline microcephaly at birth in one patient. Two of the patients exhibited behavioral anomalies including hyperactivity and aggression. Published functional data suggest a neurodevelopmental role for HIVEP2, and several of the genes regulated by HIVEP2 are implicated in brain development, for example, SSTR-2, c-Myc, and genes of the NF-κB pathway. In addition, HIVEP2-knockout mice exhibit several working memory deficits, increased anxiety, and hyperactivity. On the basis of the genotype–phenotype correlation and existing functional data, we propose HIVEP2 as a causative ID gene.

Genotype and phenotype spectrum of NRAS germline variants

RASopathies comprise a group of disorders clinically characterized by short stature, heart defects, facial dysmorphism, and varying degrees of intellectual disability and cancer predisposition. They are caused by germline variants in genes encoding key components or modulators of the highly conserved RAS-MAPK signalling pathway that lead to dysregulation of cell signal transmission. Germline changes in the genes encoding members of the RAS subfamily of GTPases are rare and associated with variable phenotypes of the RASopathy spectrum, ranging from Costello syndrome (HRAS variants) to Noonan and Cardiofaciocutaneous syndromes (KRAS variants). A small number of RASopathy cases with disease-causing germline NRAS alterations have been reported. Affected individuals exhibited features fitting Noonan syndrome, and the observed germline variants differed from the typical oncogenic NRAS changes occurring as somatic events in tumours. Here we describe 19 new cases with RASopathy due to disease-causing variants in NRAS. Importantly, four of them harbored missense changes affecting Gly12, which was previously described to occur exclusively in cancer. The phenotype in our cohort was variable but well within the RASopathy spectrum. Further, one of the patients (c.35G>A; p.(Gly12Asp)) had a myeloproliferative disorder, and one subject (c.34G>C; p.(Gly12Arg)) exhibited an uncharacterized brain tumour. With this report, we expand the genotype and phenotype spectrum of RASopathy-associated germline NRAS variants and provide evidence that NRAS variants do not spare the cancer-associated mutation hotspots.

Multiple Small Supernumerary Marker Chromosomes Resulting from Maternal Meiosis I or II Errors

We present 2 cases with multiple de novo supernumerary marker chromosomes (sSMCs), each derived from a different chromosome. In a prenatal case, we found mosaicism for an sSMC(4), sSMC(6), sSMC(9), sSMC(14) and sSMC(22), while a postnatal case had an sSMC(4), sSMC(8) and an sSMC(11). SNP-marker segregation indicated that the sSMC(4) resulted from a maternal meiosis II error in the prenatal case. Segregation of short tandem repeat markers on the sSMC(8) was consistent with a maternal meiosis I error in the postnatal case. In the latter, a boy with developmental/psychomotor delay, autism, hyperactivity, speech delay, and hypotonia, the sSMC(8) was present at the highest frequency in blood. By comparison to other patients with a corresponding duplication, a minimal region of overlap for the phenotype was identified, with CHRNB3 and CHRNA6 as dosage-sensitive candidate genes. These genes encode subunits of nicotinic acetylcholine receptors (nAChRs). We propose that overproduction of these subunits leads to perturbed component stoichiometries with dominant negative effects on the function of nAChRs, as was shown by others in vitro. With the limitation that in each case only one sSMC could be studied, our findings demonstrate that different meiotic errors lead to multiple sSMCs. We relate our findings to age-related aneuploidy in female meiosis and propose that predivision sister-chromatid separation during meiosis I or II, or both, may generate multiple sSMCs.

A cryptic unbalanced translocation der(4)t(4;17)(p16.1;q25.3) identifies Wittwer syndrome as a variant of Wolf-Hirschhorn syndrome

In 1996 Wittwer et al. described a new “mental retardation” syndrome with multiple congenital anomalies (thereupon named Wittwer syndromeOMIM300421) in threemale siblings of a family (Fig. 1A–D). One of the patients had died at the age of eightmonths (Fig. 1D). In addition to severe intellectual disability the anomalies included prenatal and severe postnatal growth retardation, dysmorphic features, blindness due to microphthalmia or optic atrophy, moderate to severe hearing loss, epilepsy, delayed bonematuration, and further congenital malformations. Progressive skeletal lesions with osteoplastic and osteoclastic changes were recognized upon clinical re-examination [Wieland et al., 2002]. The karyotype appeared normal. Pedigree analysis was highly suggestive of X-linkage since three of fourmale siblings born to two carrier sisters were affected.A linkage intervalwasassigned toXp22.3byhaplotype analysis. Further investigation usingmicrosatellite andESTmarkers refined the proposed disease locus betweenDXS8095 andDXS7108 comprising 3.9–6.1Mb and showed no evidence for deletion indicative of a continuous gene deletion syndrome within this linkage interval [Wieland et al., 2002]. Recently, we had the opportunity to re-evaluate the two living affectedmales clinically aswell as genetically. Array-basedmolecular karyotyping using anAffymetrixCytoscanHDSNP-array revealed a cryptic genomic rearrangement in both patients involving deletion of about 8.4Mbon 4p16.3p16.1 andduplication of about 3.9Mbon 17q25.3 (arr[hg19] 4p16.3p16.1(68,345–8,496,780)x1,17q25.3 (77,174,428–81,041,938)x3, Fig. 1E). Fluorescence in situ hybridization (FISH) confirmed the array results and identified the derivative chromosome der(4)t(4;17) in the patients (Fig. 1F) and the balanced translocation in both female carriers. Deletions involving chromosome region 4p16.3 cause WolfHirschhorn syndrome (WHS, OMIM 194190) [Battaglia et al., 2001]. In fact, the key clinical features of our patients meet the description of WHS including variable additional manifestations that may be explained in part by the size of the deletion in 4p16.3 [Wieczorek et al., 2000a; Maas et al., 2008]. To our knowledge, duplication of 17q25.3 in combination with 4p16.3 deletion has not beendescribed inWHS. In the sparse reports on17q25.3duplication in DECIPHER—and ISCA databases developmental delay but no specific congenitalmalformationsordysmorphic features have been described.While the partial trisomy 17q25may have contributed to