Wilhelmina S. Kerstjens-Frederikse

Nine patients with a microdeletion 15q11.2 between breakpoints 1 and 2 of the Prader–Willi critical region, possibly associated with behavioural disturbances

Behavioural differences have been described in patients with type I deletions (between breakpoints 1 and 3 (BP1-BP3)) or type II deletions (between breakpoints 2 and 3) of the 15q11.2 Prader-Willi/Angelman region. The larger type I deletions appear to coincide with more severe behavioural problems (autism, ADHD, obsessive-compulsive disorder). The non-imprinted chromosomal segment between breakpoints 1 and 2 involves four highly conserved genes, TUBGCP5, NIPA1, NIPA2, and CYFIP1; the latter three are widely expressed in the central nervous system, while TUBGCP5 is expressed in the subthalamic nuclei. These genes might explain the more severe behavioural problems seen in type I deletions. We describe nine cases with a microdeletion at 15q11.2 between BP1-BP2, thus having a haploinsufficiency for TUBGCP5, NIPA1, NIPA2, and CYFIP1 without Prader-Willi/Angelman syndrome. The clinical significance of a pure BP1-BP2 microdeletion has been debated, however, our patients shared several clinical features, including delayed motor and speech development, dysmorphisms and behavioural problems (ADHD, autism, obsessive-compulsive behaviour). Although the deletion often appeared to be inherited from a normal or mildly affected parent, it was de novo in two cases and we did not find it in 350 healthy unrelated controls. Our results suggest a pathogenic nature for the BP1-BP2 microdeletion and, although there obviously is an incomplete penetrance, they support the existence of a novel microdeletion syndrome in 15q11.2.

MLL2 mutation spectrum in 45 patients with Kabuki syndrome

Kabuki Syndrome (KS) is a rare syndrome characterized by intellectual disability and multiple congenital abnormalities, in particular a distinct dysmorphic facial appearance. KS is caused by mutations in the MLL2 gene, encoding an H3K4 histone methyl transferase which acts as an epigenetic transcriptional activator during growth and development. Direct sequencing of all 54 exons of the MLL2 gene in 45 clinically well‐defined KS patients identified 34 (75.6%) different mutations. One mutation has been described previously, all others are novel. Clinically, all KS patients were sporadic, and mutations were de novo for all 27 families for which both parents were available. We detected nonsense (n=11), frameshift (n=17), splice site (n=4) and missense (n=2) mutations, predicting a high frequency of absent or non‐functional MLL2 protein. Interestingly, both missense mutations located in the C‐terminal conserved functional domains of the protein. Phenotypically our study indicated a statistically significant difference in the presence of a distinct facial appearance (p=0.0143) and growth retardation (p=0.0040) when comparing KS patients with an MLL2 mutation compared to patients without a mutation. Our data double the number of MLL2 mutations in KS reported so far and widen the spectrum of MLL2 mutations and disease mechanisms in KS.

Clinical heterogeneity in lymphoedema-distichiasis with FOXC2 truncating mutations

BACKGROUND Hereditary lymphoedema-distichiasis (LD) is an autosomal dominant disorder that classically presents as lymphoedema of the limbs, with variable age of onset, and extra aberrant growth of eyelashes from the Meibomian gland (distichiasis). Other major reported complications include cardiac defects, cleft palate, and extradural cysts. Photophobia, exotropia, ptosis, congenital ectropion, and congenital cataracts are additional eye findings. Recently, we reported that truncating mutations in the forkhead transcription family member FOXC2resulted in LD in two families. METHODS The clinical findings in seven additional families with LD, including the original family described by Falls and Kertesz, were determined and mutational analyses were performed. RESULTS Distichiasis was the most common clinical feature followed by age dependent lymphoedema. There is a wide variation of associated secondary features including tetralogy of Fallot and cleft palate. The mutational analyses identified truncating mutations in all of the families studied (two nonsense, one deletion, three insertion, and one insertion-deletion), which most likely result in haploinsufficiency ofFOXC2. CONCLUSIONS FOXC2mutations are highly penetrant with variable expressivity which is not explicable by the pattern of mutations.

First-trimester use of paroxetine and congenital heart defects: A population-based case-control study†‡

BACKGROUND There is a need for case-control studies of the effect of paroxetine on the occurrence of specific heart defects. METHODS We performed a case-control study with data from a population-based birth defects registry in the Netherlands. All the children born between 1997 and 2006 were selected. Cases were defined as fetuses and children with isolated heart defects, and the controls were fetuses and children with a genetic disorder with no heart defect. We excluded children for whom there was no information on maternal medication use and deceased children and fetuses who were not examined postmortem. First-trimester exposure to paroxetine was compared between cases and controls by calculating adjusted odds ratios (AOR). RESULTS We included 678 cases with isolated heart defects and 615 controls. The first trimester exposure rate was 1.5% for cases and 1.0% for controls. After excluding mothers who used paroxetine outside the first trimester, or who had used another SSRI, we found no significantly increased risk for heart defects overall (10 exposed cases; AOR, 1.5; 95% confidence interval [CI], 0.5-4.0), but we did find a significantly increased risk for atrium septum defects (three exposed cases; AOR, 5.7; 95% CI, 1.4-23.7). CONCLUSIONS Our results suggest that the use of paroxetine in early pregnancy is associated with an increased risk of atrium septum defects. The results stress the importance of studying possible teratogenic effects of a drug, preferably in regard to well-specified malformations.

The role of maternal-fetal cholesterol transport in early fetal life: Current insights

ABSTRACT The importance of maternal cholesterol as an exogenous cholesterol source for the growing embryo was first reported in studies of Smith-Lemli-Opitz syndrome. Although most of the fetuss cholesterol is synthesized by the fetus itself, there is now growing evidence that during the first weeks of life, when most organs develop, the fetus largely depends on maternal cholesterol as its cholesterol source. The maternal-fetal cholesterol transport mechanism, by transporters in both the yolk sac and placenta, is becoming better understood. This minireview summarizes current insights on maternal-fetal cholesterol transport based on in vitro and in vivo studies. As the prevalence of maternal diseases, such as diabetes, obesity, and the metabolic syndrome that adversely affect maternal cholesterol levels, is now rapidly reaching epidemic proportions, we urgently need to determine the impact of these maternal conditions on the developing human fetus.

TBX4 mutations (small patella syndrome) are associated with childhood-onset pulmonary arterial hypertension

Background Childhood-onset pulmonary arterial hypertension (PAH) is rare and differs from adult-onset disease in clinical presentation, with often unexplained mental retardation and dysmorphic features (MR/DF). Mutations in the major PAH gene, BMPR2, were reported to cause PAH in only 10–16% of childhood-onset patients. We aimed to identify more genes associated with childhood-onset PAH. Methods We studied 20 consecutive cases with idiopathic or heritable PAH. In patients with accompanying MR/DF (n=6) array-comparative genomic hybridisation analysis was performed, with the aim of finding common deletion regions containing candidate genes for PAH. Three patients had overlapping deletions of 17q23.2. TBX2 and TBX4 were selected from this area as candidate genes and sequenced in all 20 children. After identifying TBX4 mutations in these children, we subsequently sequenced TBX4 in a cohort of 49 adults with PAH. Because TBX4 mutations are known to cause small patella syndrome (SPS), all patients with newly detected TBX4 mutations were screened for features of SPS. We also screened a third cohort of 23 patients with SPS for PAH. Results TBX4 mutations (n=3) or TBX4-containing deletions (n=3) were detected in 6 out of 20 children with PAH (30%). All living patients and two parents with TBX4 mutations appeared to have previously unrecognised SPS. In the adult PAH-cohort, one TBX4 mutation (2%) was detected. Screening in the cohort of (predominantly adult) SPS patients revealed no PAH. Conclusions These data indicate that TBX4 mutations are associated with childhood-onset PAH, but that the prevalence of PAH in adult TBX4 mutation carriers is low.

Left ventricular outflow tract obstruction: should cardiac screening be offered to first-degree relatives?

Objectives To determine whether offering cardiac screening to relatives of patients with left ventricular outflow tract obstructions (LVOTOs) would be justified. Background LVOTOs have been recognised as a group of congenital heart diseases with ‘high heritability’. One of the LVOTOs, the bicuspid aortic valve, is often asymptomatic, but has become known to be associated with sudden, unexpected cardiac death. However, the need for cardiac screening of first-degree relatives of patients with LVOTO has not been determined owing to the lack of studies in well-defined cohorts of consecutive patients. Methods The families of a cohort of 249 consecutive paediatric patients with LVOTO were offered genetic counselling. Of 182 consenting index patients, 40 patients (22%) appeared to have associated non-cardiac congenital anomalies (LVOTO-NCA). In the other 142 patients with LVOTO, cardiac screening of 449 first-degree relatives was performed. Results Cardiac screening disclosed a cardiac anomaly in 34 first-degree relatives (8%). In 23 (68%) of these the cardiac anomaly was a bicuspid aortic valve. Twenty-four of these anomalies were newly detected by our screening programme (71%). These 34 cardiac anomalies were found in the families of 28 index cases (20%). Conclusions This study shows that of the patients with LVOTO without NCA, 20% had (an) affected first-degree relative(s), frequently with undetected bicuspid aortic valves. These data suggest that cardiac screening of relatives of patients with LVOTO without NCA is justified. This may help prevent sudden, unexpected, cardiac death or life-threatening complications in relatives with undetected bicuspid aortic valves.

More Clinical Overlap between 22q11.2 Deletion Syndrome and CHARGE Syndrome than Often Anticipated

CHARGE (coloboma, heart defects, atresia of choanae, retardation of growth and development, genital hypoplasia, and ear abnormalities) and 22q11.2 deletion syndromes are variable, congenital malformation syndromes that show considerable phenotypic overlap. We further explored this clinical overlap and proposed recommendations for the genetic diagnosis of both syndromes. We described 2 patients clinically diagnosed with CHARGE syndrome, who were found to carry a 22q11.2 deletion, and searched the literature for more cases. In addition, we screened our cohort of CHD7 mutation carriers (n = 802) for typical 22q11.2 deletion features and studied CHD7 in 20 patients with phenotypically 22q11.2 deletion syndrome but without haploinsufficiency of TBX1. In total, we identified 5 patients with a clinical diagnosis of CHARGE syndrome and a proven 22q11.2 deletion. Typical 22q11.2 deletion features were found in 30 patients (30/802, 3.7%) of our CHD7 mutation-positive cohort. We found truncating CHD7 mutations in 5/20 patients with phenotypically 22q11.2 deletion syndrome. Differentiating between CHARGE and 22q11.2 deletion syndromes can be challenging. CHD7 and TBX1 probably share a molecular pathway or have common target genes in affected organs. We strongly recommend performing CHD7 analysis in patients with a 22q11.2 deletion phenotype without TBX1 haploinsufficiency and conversely, performing a genome-wide array in CHARGE syndrome patients without a CHD7 mutation.

Cardiovascular malformations caused by NOTCH1 mutations do not keep left: data on 428 probands with left-sided CHD and their families

Purpose:We aimed to determine the prevalence and phenotypic spectrum of NOTCH1 mutations in left-sided congenital heart disease (LS-CHD). LS-CHD includes aortic valve stenosis, a bicuspid aortic valve, coarctation of the aorta, and hypoplastic left heart syndrome.Methods:NOTCH1 was screened for mutations in 428 nonsyndromic probands with LS-CHD, and family histories were obtained for all. When a mutation was detected, relatives were also tested.Results:In 148/428 patients (35%), LS-CHD was familial. Fourteen mutations (3%; 5 RNA splicing mutations, 8 truncating mutations, 1 whole-gene deletion) were detected, 11 in familial disease (11/148 (7%)) and 3 in sporadic disease (3/280 (1%)). Forty-nine additional mutation carriers were identified among the 14 families, of whom 12 (25%) were asymptomatic. Most of these mutation carriers had LS-CHD, but 9 (18%) had right-sided congenital heart disease (RS-CHD) or conotruncal heart disease (CTD). Thoracic aortic aneurysms (TAAs) occurred in 6 mutation carriers (probands included 6/63 (10%)).Conclusion:Pathogenic mutations in NOTCH1 were identified in 7% of familial LS-CHD and in 1% of sporadic LS-CHD. The penetrance is high; a cardiovascular malformation was found in 75% of NOTCH1 mutation carriers. The phenotypic spectrum includes LS-CHD, RS-CHD, CTD, and TAA. Testing NOTCH1 for an early diagnosis in LS-CHD/RS-CHD/CTD/TAA is warranted.Genet Med 18 9, 914–923.

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.