Hermine E. Veenstra-Knol

Severe cardiac phenotype with right ventricular predominance in a large cohort of patients with a single missense mutation in the DES gene

BACKGROUND Desmin-related myopathy is a clinically heterogenous group of disorders encompassing myopathies, cardiomyopathies, conduction disease, and combinations of these disorders. Mutations in the gene encoding desmin (DES), a major intermediate filament protein, can underlie this phenotype. OBJECTIVE The purpose of this study was to investigate the clinical and pathologic characteristics of 27 patients from five families with an identical mutation in the head domain region (p.S13F) of desmin. METHODS/RESULTS All 27 carriers or obligate carriers of a p.S13F DES founder mutation demonstrated a fully penetrant yet variable phenotype. All patients demonstrated cardiac involvement characterized by high-grade AV block at young ages and important right ventricular (RV) involvement. RV predominance was demonstrated by the presence of right bundle branch block in 10 patients (sometimes as a first manifestation) and by RV heart failure in 6 patients, including 2 patients who fulfilled the diagnostic criteria for arrhythmogenic RV cardiomyopathy. Because of this clinical overlap with desmosome cardiomyopathies, we also studied the organization of the intercalated disks, particularly the distribution of desmosomal proteins. Normal amounts of the major desmosomal proteins were found, but the intercalated disks were more convoluted and elongated and had a zigzag appearance. CONCLUSION In this largest series to date of individuals with a single head domain DES mutation, patients show a variable yet predominantly cardiologic phenotype characterized by conduction disease at an early age and RV involvement including right bundle branch block and/or RV tachycardias and arrhythmogenic RV cardiomyopathy phenocopies. A localized effect of desmin on the structure of the cardiac intercalated disks might contribute to disease pathogenesis.

Paediatric intestinal cancer and polyposis due to bi-allelic PMS2 mutations: Case series, review and follow-up guidelines

BACKGROUND Bi-allelic germline mutations of one of the DNA mismatch repair genes, so far predominantly found in PMS2, cause constitutional MMR-deficiency syndrome. This rare disorder is characterised by paediatric intestinal cancer and other malignancies. We report the clinical, immunohistochemical and genetic characterisation of four families with bi-allelic germline PMS2 mutations. We present an overview of the published gastrointestinal manifestations of CMMR-D syndrome and propose recommendations for gastro-intestinal screening. METHODS AND RESULTS The first proband developed a cerebral angiosarcoma at age 2 and two colorectal adenomas at age 7. Genetic testing identified a complete PMS2 gene deletion and a frameshift c.736_741delinsTGTGTGTGAAG (p.Pro246CysfsX3) mutation. In the second family, both the proband and her brother had multiple intestinal adenomas, initially wrongly diagnosed as familial adenomatous polyposis. A splice site c.2174+1G>A, and a missense c.137G>T (p.Ser46Ile) mutation in PMS2 were identified. The third patient was diagnosed with multiple colorectal adenomas at age 11; he developed a high-grade dysplastic colorectal adenocarcinoma at age 21. Two intragenic PMS2 deletions were found. The fourth proband developed a cerebral anaplastic ganglioma at age 9 and a high-grade colerectal dysplastic adenoma at age 10 and carries a homozygous c.2174+1G>A mutation. Tumours of all patients showed microsatellite instability and/or loss of PMS2 expression. CONCLUSIONS Our findings show the association between bi-allelic germline PMS2 mutations and severe childhood-onset gastrointestinal manifestations, and support the notion that patients with early-onset gastrointestinal adenomas and cancer should be investigated for CMMR-D syndrome. We recommend yearly follow-up with colonoscopy from age 6 and simultaneous video-capsule small bowel enteroscopy from age 8.

Genotype-Phenotype Correlation in Patients Suspected of Having Sotos Syndrome

Background: Deletions and mutations in the NSD1 gene are the major cause of Sotos syndrome. We wanted to evaluate the genotype-phenotype correlation in patients suspected of having Sotos syndrome and determine the best discriminating parameters for the presence of a NSD1 gene alteration. Methods: Mutation and fluorescence in situ hybridization analysis was performed on blood samples of 59 patients who were clinically scored into 3 groups. Clinical data were compared between patients with and without NSD1 alterations. With logistic regression analysis the best combination of predictive variables was obtained. Results: In the groups of typical, dubious and atypical Sotos syndrome, 81, 36 and 0% of the patients, respectively, showed NSD1 gene alterations. Four deletions were detected. In 23 patients (2 families) 19 mutations were detected (1 splicing defect, 3 non-sense, 7 frameshift and 8 missense mutations). The best predictive parameters for a NSD1 gene alteration were frontal bossing, down-slanted palpebral fissures, pointed chin and overgrowth. Higher incidences of feeding problems and cardiac anomalies were found. The parameters, delayed development and advanced bone age, did not differ between the 2 subgroups. Conclusions: In our patients suspected of having Sotos syndrome, facial features and overgrowth were highly predictive of a NSD1 gene aberration, whereas developmental delay and advanced bone age were not.

Mutations in NEK8 link multiple organ dysplasia with altered Hippo signalling and increased c-MYC expression

Mutations affecting the integrity and function of cilia have been identified in various genes over the last decade accounting for a group of diseases called ciliopathies. Ciliopathies display a broad spectrum of phenotypes ranging from mild manifestations to lethal combinations of multiple severe symptoms and most of them share cystic kidneys as a common feature. Our starting point was a consanguineous pedigree with three affected fetuses showing an early embryonic phenotype with enlarged cystic kidneys, liver and pancreas and developmental heart disease. By genome-wide linkage analysis, we mapped the disease locus to chromosome 17q11 and identified a homozygous nonsense mutation in NEK8/NPHP9 that encodes a kinase involved in ciliary dynamics and cell cycle progression. Missense mutations in NEK8/NPHP9 have been identified in juvenile cystic kidney jck mice and in patients suffering from nephronophthisis (NPH), an autosomal-recessive cystic kidney disease. This work confirmed a complete loss of NEK8 expression in the affected fetuses due to nonsense-mediated decay. In cultured fibroblasts derived from these fetuses, the expression of prominent polycystic kidney disease genes (PKD1 and PKD2) was decreased, whereas the oncogene c-MYC was upregulated, providing potential explanations for the observed renal phenotype. We furthermore linked NEK8 with NPHP3, another NPH protein known to cause a very similar phenotype in case of null mutations. Both proteins interact and activate the Hippo effector TAZ. Taken together, our study demonstrates that NEK8 is essential for organ development and that the complete loss of NEK8 perturbs multiple signalling pathways resulting in a severe early embryonic phenotype.

Functional analysis of novel TBX5 T-box mutations associated with Holt–Oram syndrome

AIMS Holt-Oram syndrome (HOS) is a heart/hand syndrome clinically characterized by upper limb and cardiac malformations. Mutations in T-box transcription factor 5 (TBX5) underlie this syndrome, the majority of which lead to premature stops. In this study, we present our functional analyses of five (novel) missense TBX5 mutations identified in HOS patients, most of whom presented with severe cardiac malformations. METHODS AND RESULTS Functional characterization of mutant proteins shows a dramatic loss of DNA-binding capacity, as well as diminished binding to known cardiac interaction partners NKX2-5 and GATA4. The disturbance of these interactions leads to a loss of function, as measured by the reduced activation of Nppa and FGF10 in rat heart derived cells, although with variable severity. Two out of the five mutations are peculiar: one, p.H220del, is associated with additional extra-cardiac defects, perhaps by interfering with other T-box dependant pathways, and another, p.I106V, leads to limb defects only, which is supported by its normal interaction with cardiac-specific interaction partners. CONCLUSION Overall, our data are consistent with the hypothesis that these novel missense mutations in TBX5 lead to functional haploinsufficiency and result in a reduced transcriptional activation of target genes, which is likely central to the pathogenesis of HOS.

Practical guidelines for interpreting copy number gains detected by high-resolution array in routine diagnostics

The correct interpretation of copy number gains in patients with developmental delay and multiple congenital anomalies is hampered by the large number of copy number variations (CNVs) encountered in healthy individuals. The variable phenotype associated with copy number gains makes interpretation even more difficult. Literature shows that inheritence, size and presence in healthy individuals are commonly used to decide whether a certain copy number gain is pathogenic, but no general consensus has been established. We aimed to develop guidelines for interpreting gains detected by array analysis using array CGH data of 300 patients analysed with the 105K Agilent oligo array in a diagnostic setting. We evaluated the guidelines in a second, independent, cohort of 300 patients. In the first 300 patients 797 gains of four or more adjacent oligonucleotides were observed. Of these, 45.4% were de novo and 54.6% were familial. In total, 94.8% of all de novo gains and 87.1% of all familial gains were concluded to be benign CNVs. Clinically relevant gains ranged from 288 to 7912 kb in size, and were significantly larger than benign gains and gains of unknown clinical relevance (P<0.001). Our study showed that a threshold of 200 kb is acceptable in a clinical setting, whereas heritability does not exclude a pathogenic nature of a gain. Evaluation of the guidelines in the second cohort of 300 patients revealed that the interpretation guidelines were clear, easy to follow and efficient.

A Study of the Clinical and Radiological Features in a Cohort of 93 Patients with a COL2A1 Mutation Causing Spondyloepiphyseal Dysplasia Congenita or a Related Phenotype

Type 2 collagen disorders encompass a diverse group of skeletal dysplasias that are commonly associated with orthopedic, ocular, and hearing problems. However, the frequency of many clinical features has never been determined. We retrospectively investigated the clinical, radiological, and genotypic data in a group of 93 patients with molecularly confirmed SEDC or a related disorder. The majority of the patients (80/93) had short stature, with radiological features of SEDC (n = 64), others having SEMD (n = 5), Kniest dysplasia (n = 7), spondyloperipheral dysplasia (n = 2), or Torrance‐like dysplasia (n = 2). The remaining 13 patients had normal stature with mild SED, Stickler‐like syndrome or multiple epiphyseal dysplasia. Over 50% of the patients had undergone orthopedic surgery, usually for scoliosis, femoral osteotomy or hip replacement. Odontoid hypoplasia was present in 56% (95% CI 38–74) and a correlation between odontoid hypoplasia and short stature was observed. Atlanto‐axial instability, was observed in 5 of the 18 patients (28%, 95% CI 10–54) in whom flexion‐extension films of the cervical spine were available; however, it was rarely accompanied by myelopathy. Myopia was found in 45% (95% CI 35–56), and retinal detachment had occurred in 12% (95% CI 6–21; median age 14 years; youngest age 3.5 years). Thirty‐two patients complained of hearing loss (37%, 95% CI 27–48) of whom 17 required hearing aids. The ophthalmological features and possibly also hearing loss are often relatively frequent and severe in patients with splicing mutations. Based on clinical findings, age at onset and genotype–phenotype correlations in this cohort, we propose guidelines for the management and follow‐up in this group of disorders.

The Koolen-de Vries syndrome: A phenotypic comparison of patients with a 17q21.31 microdeletion versus a KANSL1 sequence variant

The Koolen-de Vries syndrome (KdVS; OMIM #610443), also known as the 17q21.31 microdeletion syndrome, is a clinically heterogeneous disorder characterised by (neonatal) hypotonia, developmental delay, moderate intellectual disability, and characteristic facial dysmorphism. Expressive language development is particularly impaired compared with receptive language or motor skills. Other frequently reported features include social and friendly behaviour, epilepsy, musculoskeletal anomalies, congenital heart defects, urogenital malformations, and ectodermal anomalies. The syndrome is caused by a truncating variant in the KAT8 regulatory NSL complex unit 1 (KANSL1) gene or by a 17q21.31 microdeletion encompassing KANSL1. Herein we describe a novel cohort of 45 individuals with KdVS of whom 33 have a 17q21.31 microdeletion and 12 a single-nucleotide variant (SNV) in KANSL1 (19 males, 26 females; age range 7 months to 50 years). We provide guidance about the potential pitfalls in the laboratory testing and emphasise the challenges of KANSL1 variant calling and DNA copy number analysis in the complex 17q21.31 region. Moreover, we present detailed phenotypic information, including neuropsychological features, that contribute to the broad phenotypic spectrum of the syndrome. Comparison of the phenotype of both the microdeletion and SNV patients does not show differences of clinical importance, stressing that haploinsufficiency of KANSL1 is sufficient to cause the full KdVS phenotype.

Further delineation of the KAT6B molecular and phenotypic spectrum

KAT6B sequence variants have been identified previously in both patients with the Say-Barber-Biesecker type of blepharophimosis mental retardation syndromes (SBBS) and in the more severe genitopatellar syndrome (GPS). We report on the findings in a previously unreported group of 57 individuals with suggestive features of SBBS or GPS. Likely causative variants have been identified in 34/57 patients and were commonly located in the terminal exons of KAT6B. Of those where parental samples could be tested, all occurred de novo. Thirty out of thirty-four had truncating variants, one had a missense variant and the remaining three had the same synonymous change predicted to affect splicing. Variants in GPS tended to occur more proximally to those in SBBS patients, and genotype/phenotype analysis demonstrated significant clinical overlap between SBBS and GPS. The de novo synonymous change seen in three patients with features of SBBS occurred more proximally in exon 16. Statistical analysis of clinical features demonstrated that KAT6B variant-positive patients were more likely to display hypotonia, feeding difficulties, long thumbs/great toes and dental, thyroid and patella abnormalities than KAT6B variant-negative patients. The few reported patients with KAT6B haploinsufficiency had a much milder phenotype, though with some features overlapping those of SBBS. We report the findings in a previously unreported patient with a deletion of the KAT6B gene to further delineate the haploinsufficiency phenotype. The molecular mechanisms giving rise to the SBBS and GPS phenotypes are discussed.

Central 22q11.2 deletions

22q11.2 deletion syndrome is one of the most common microdeletion syndromes. Most patients have a deletion resulting from a recombination of low copy repeat blocks LCR22‐A and LCR22‐D. Loss of the TBX1 gene is considered the most important cause of the phenotype. A limited number of patients with smaller, overlapping deletions distal to the TBX1 locus have been described in the literature. In these patients, the CRKL gene is deleted. Haploinsufficiency of this gene has also been implicated in the pathogenesis of 22q11.2 deletion syndrome. To distinguish these deletions (comprising the LCR22‐B to LCR22‐D region) from the more distal 22q11.2 deletions (located beyond LCR22‐D), we propose the term “central 22q11.2 deletions”. In the present study we report on 27 new patients with such a deletion. Together with information on previously published cases, we review the clinical findings of 52 patients. The prevalence of congenital heart anomalies and the frequency of de novo deletions in patients with a central deletion are substantially lower than in patients with a common or distal 22q11.2 deletion. Renal and urinary tract malformations, developmental delays, cognitive impairments and behavioral problems seem to be equally frequent as in patients with a common deletion. None of the patients had a cleft palate. Patients with a deletion that also encompassed the MAPK1 gene, located just distal to LCR22‐D, have a different and more severe phenotype, characterized by a higher prevalence of congenital heart anomalies, growth restriction and microcephaly. Our results further elucidate genotype‐phenotype correlations in 22q11.2 deletion syndrome spectrum.