Innovative all-in-one exome sequencing strategy for diagnostic genetic testing in male infertility: Validation and 10-month experience.

BACKGROUND: Current guidelines indicate that patients with extreme oligozoospermia or azoospermia should be tested for chromosomal imbalances, azoospermia factor (AZF) deletions and/or CFTR variants. For other sperm abnormalities, no genetic diagnostics are recommended. OBJECTIVES: To determine whether exome sequencing (ES) with combined copy number variant (CNV) and single nucleotide variant (SNV) analysis is a reliable first-tier method to replace current methods (validation study), and to evaluate the diagnostic yield after 10 months of implementation (evaluation study). MATERIALS AND METHODS: In the validation study, ES was performed on DNA of patients already diagnosed with AZF deletions (n = 17), (non-)mosaic sex chromosomal aneuploidies or structural chromosomal anomalies (n = 37), CFTR variants (n = 26), or variants in known infertility genes (n = 4), and 90 controls. The data were analyzed using our standard diagnostic pipeline, with a bioinformatic filter for 130 male infertility genes. In the evaluation study, results of 292 clinical exomes were included. RESULTS: All previously reported variants in the validation cohort, including clinically relevant Y-chromosomal microdeletions, were correctly identified and reliably detected. In the evaluation study, we identified one or more clinically relevant genetic anomalies in 67 of 292 of all cases (22.9%): these included aberrations that could have been detected with current methods in 30 of 67 patients (10.2% of total), (possible) (mono)genetic causes in the male infertility gene panel in 28 of 67 patients (9.6%), and carriership of cystic fibrosis in nine of 67 patients (3.1%). CONCLUSION: ES is a reliable first-tier method to detect the most common genetic causes of male infertility and, as additional genetic causes can be detected, in our evaluation cohort the diagnostic yield almost doubled (10.2%-19.8%, excluding CF carriers). A genetic diagnosis provides answers on the cause of infertility and helps the professionals in the counseling for treatment, possible co-morbidities and risk for offspring and/or family members. Karyotyping will still remain necessary for detecting balanced translocations or low-grade chromosomal mosaicism.

Vascular Ehlers-Danlos Syndrome: A Comprehensive Natural History Study in a Dutch National Cohort of 142 Patients.

BACKGROUND: Vascular Ehlers-Danlos syndrome (vEDS) is a rare connective tissue disorder with a high risk for arterial, bowel, and uterine rupture, caused by heterozygous pathogenic variants in COL3A1. The aim of this cohort study is to provide further insights into the natural history of vEDS and describe genotype-phenotype correlations in a Dutch multicenter cohort to optimize patient care and increase awareness of the disease. METHODS: Individuals with vEDS throughout the Netherlands were included. The phenotype was charted by retrospective analysis of molecular and clinical data, combined with a one-time physical examination. RESULTS: A total of 142 individuals (50% female) participated the study, including 46 index patients (32%). The overall median age at genetic diagnosis was 41.0 years. More than half of the index patients (54.3%) and relatives (53.1%) had a physical appearance highly suggestive of vEDS. In these individuals, major events were not more frequent (P=0.90), but occurred at a younger age (P=0.01). A major event occurred more often and at a younger age in men compared with women (P<0.001 and P=0.004, respectively). Aortic aneurysms (P=0.003) and pneumothoraces (P=0.029) were more frequent in men. Aortic dissection was more frequent in individuals with a COL3A1 variant in the first quarter of the collagen helical domain (P=0.03). CONCLUSIONS: Male sex, type and location of the COL3A1 variant, and physical appearance highly suggestive of vEDS are risk factors for the occurrence and early age of onset of major events. This national multicenter cohort study of Dutch individuals with vEDS provides a valuable basis for improving guidelines for the diagnosing, follow-up, and treatment of individuals with vEDS.

Type A Aortic Dissection in a 24-Year-Old Patient With Kabuki Syndrome.

Our case report documents the first type A aortic dissection in a patient with Kabuki syndrome (KS) and emphasize the need for intensive cardiovascular risk monitoring in patients with KS. It stresses the importance of further research to establish a correlation and awareness for patients with KS.

Comparability of different Z-score equations for aortic root dimensions in children with Marfan syndrome.

BACKGROUND: Aortic root dilation is a major complication of Marfan syndrome and is one of the most important criteria in establishing the diagnosis. Currently, different echocardiographic nomograms are used to calculate aortic root Z-scores. The aim of the present study was to assess the potential differences in aortic root measurements when aortic root Z-scores were obtained in a cohort of paediatric Marfan patients using several published nomograms. METHODS: In a cohort of 100 children with Marfan syndrome, Z-scores for aortic root dimensions were calculated according to the nomograms of Pettersen et al, Gautier et al, Colan et al, and Lopez et al. Bland-Altman plots were used to estimate mean differences in Z-scores and to establish limits of agreement. RESULTS: The mean Z-score of the sinus of Valsalva for Lopez et al was significantly higher compared to Gautier et al (p < 0.01) and Pettersen et al (p = 0.03). The nomogram of Lopez et al resulted in substantially higher Z-scores in patients with a large sinus of Valsalva diameter. Thirty-five percentage of the studied patients would have a Z-score ≥ 2 using Lopez et al compared to 20% for Pettersen et al, 21% for Gautier et al, and 33% for Colan et al. CONCLUSION: The currently available nomograms for calculating Z-scores of aortic dilation in children with Marfan syndrome lead to clinically relevant differences in Z-scores, especially in children with a relative large aortic root diameter. This could have impact on both the diagnosis and treatment of patients with Marfan syndrome.

Germline and somatic mosaicism in a family with multiple endocrine neoplasia type 1 (MEN1) syndrome.

Context Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disease caused by mutations in the tumor suppressor gene MEN1 and can be diagnosed based on clinical, familial and/or genetic criteria. We present a family in which we found both germline and somatic mosaicism for MEN1. Family description In our proband, we diagnosed MEN1. The mutation was not detected in her parents (DNA extracted from leucocytes). When her brother was found to harbor the same MEN1 mutation as our proband and, around the same time, their father was diagnosed with a neuroendocrine carcinoma, this tumor was investigated for the MEN1 mutation as well. In the histologic biopsy of this tumor, the same MEN1 mutation was detected as previously found in his children. Re-analysis of his blood using multiplex ligation-dependent probe amplification (MLPA) showed a minimal, but consistently decreased signal for the MEN1-specific MLPA probes. The deletion was confirmed in his son by high-resolution array analysis. Based on the array data, we concluded that the deletion was limited to the MEN1 gene and that the father had both germline and somatic mosaicism for MEN1. Conclusions To our knowledge, this is the first reported family with combined germline and somatic mosaicism for MEN1. This study illustrates that germline mosaicism is important to consider in apparently sporadic de novo MEN1 mutations, because of its particular importance for genetic counseling, specifically when evaluating the risk for family members and when considering the possibility of somatic mosaicism in the parent with germline mosaicism.

Mutations in N-acetylglucosamine (O-GlcNAc) transferase in patients with X-linked intellectual disability.

N-Acetylglucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential and dynamic post-translational modification. The O-GlcNAc modification is present on numerous nuclear and cytosolic proteins and has been implicated in essential cellular functions such as signaling and gene expression. Accordingly, altered levels of protein O-GlcNAcylation have been associated with developmental defects and neurodegeneration. However, mutations in the OGT gene have not yet been functionally confirmed in humans. Here, we report on two hemizygous mutations in OGT in individuals with X-linked intellectual disability (XLID) and dysmorphic features: one missense mutation (p.Arg284Pro) and one mutation leading to a splicing defect (c.463-6T>G). Both mutations reside in the tetratricopeptide repeats of OGT that are essential for substrate recognition. We observed slightly reduced levels of OGT protein and reduced levels of its opposing enzyme O-GlcNAcase in both patient-derived fibroblasts, but global O-GlcNAc levels appeared to be unaffected. Our data suggest that mutant cells attempt to maintain global O-GlcNAcylation by down-regulating O-GlcNAcase expression. We also found that the c.463-6T>G mutation leads to aberrant mRNA splicing, but no stable truncated protein was detected in the corresponding patient-derived fibroblasts. Recombinant OGT bearing the p.Arg284Pro mutation was prone to unfolding and exhibited reduced glycosylation activity against a complex array of glycosylation substrates and proteolytic processing of the transcription factor host cell factor 1, which is also encoded by an XLID-associated gene. We conclude that defects in O-GlcNAc homeostasis and host cell factor 1 proteolysis may play roles in mediation of XLID in individuals with OGT mutations.

[Neonatal screening for congenital hypothyroidism: more than 30 years of experience in the Netherlands]. / Neonatale screening op congenitale hypothyreoïdie: ruim 30 jaar ervaring in Nederland.

OBJECTIVE: To describe the Dutch neonatal screening programme for congenital hypothyroidism (CH). DESIGN: Descriptive study. METHOD: Data on neonatal screening for CH in the period 1 January 1981 through 31 December 2011 were obtained from the Department for Vaccine Supply and Prevention Programmes of the Dutch National Institute for Public Health and the Environment (RIVM), laboratories and paediatricians to whom babies with abnormal screening results were referred. The screening procedure has been amended several times. In the period 1981-1994, only T4 and TSH were measured in heel prick blood, for example. From 1995, thyroxine-binding globulin (TBG) was added to the screening protocol. RESULTS: The participation rate was 99.7%. Before 1995 the sensitivity, specificity and positive predictive value were 94%, 99.51% and 6%, respectively. From 1995 these percentages were 98%, 99.85% and 21%, respectively. The total prevalence of CH was 1:2670 (prevalence of CH of thyroidal origin was 1:3100 and CH of central origin was 1:21,600). The percentages of patients with severe CH treated before day 15 in the periods 1981-1990, 1991-2000 and 2001-2011 were 24% (63/263), 63% (170/269) and 96% (176/184), respectively. CONCLUSION: The sensitivity and specificity of the screening procedure has considerably increased since 1995 compared with the period before 1995. In recent years patients with severe CH were treated considerably earlier than in the first years of the screening. Neonatal screening for CH may be considered as an important success for public health care.

A dominant-negative GFI1B mutation in the gray platelet syndrome.

The gray platelet syndrome is a hereditary, usually autosomal recessive bleeding disorder caused by a deficiency of alpha granules in platelets. We detected a nonsense mutation in the gene encoding the transcription factor GFI1B (growth factor independent 1B) that causes autosomal dominant gray platelet syndrome. Both gray platelets and megakaryocytes had abnormal marker expression. In addition, the megakaryocytes had dysplastic features, and they were abnormally distributed in the bone marrow. The GFI1B mutant protein inhibited nonmutant GFI1B transcriptional activity in a dominant-negative manner. Our studies show that GFI1B, in addition to being causally related to the gray platelet syndrome, is key to megakaryocyte and platelet development.

Evaluation of cognitive and motor development in toddlers with congenital hypothyroidism diagnosed by neonatal screening.

OBJECTIVE: The Dutch neonatal congenital hypothyroidism (CH) screening procedure and treatment modality has been adapted several times since its national institution in 1981. These changes enabled us to investigate whether earlier treatment has resulted in improved cognitive and motor outcomes. The present study examined whether the advancement of treatment modality has resulted in improved cognitive and motor outcomes. METHODS: In 95 toddlers with thyroidal CH (CH-T), born in 2002 through 2004 and treated at a median age of 9 days, cognitive and motor outcomes were assessed with the Bayley Scales of Infant Development-II-NL at 1 and 2 years of age. This outcome was also analyzed in relation to treatment variables. RESULTS: The mean mental developmental index (MDI) scores of the severe (initial free thyroxine [FT4] ≤0.4 ng/dL (≤5 pmol/L), moderate (0.4 < initial FT4 ≤ 0.8 ng/dL (5.0 < initial FT4 ≤ 10.0 pmol/L), and mild (initial FT4 > 0.8 ng/dL (>10.0 pmol/l) CH-T groups at 1 year and the moderate and mild CH-T groups at 2 years were similar to the population mean. The mean MDI scores of the total CH-T group and severe CH-T group at 2 years were significantly lower than the population mean (p < .0001). In all 3 severity subgroups, significant lower psychomotor developmental index scores (p < .0001) were observed. No correlations were found between starting day of treatment and developmental outcome. Initial T4 concentration and initial T4 dose were weak predictors for developmental outcome. CONCLUSION: Essentially, comparable with our earlier findings, children with CH, especially those with severe CH, are still at risk for motor and cognitive problems, which are probably due to the consequence of the prenatal hypothyroid state or the thyroid hormone deficiency in early life.

Loss-of-function mutations in TGFB2 cause a syndromic presentation of thoracic aortic aneurysm.

Loeys-Dietz syndrome (LDS) associates with a tissue signature for high transforming growth factor (TGF)-ß signaling but is often caused by heterozygous mutations in genes encoding positive effectors of TGF-ß signaling, including either subunit of the TGF-ß receptor or SMAD3, thereby engendering controversy regarding the mechanism of disease. Here, we report heterozygous mutations or deletions in the gene encoding the TGF-ß2 ligand for a phenotype within the LDS spectrum and show upregulation of TGF-ß signaling in aortic tissue from affected individuals. Furthermore, haploinsufficient Tgfb2(+/-) mice have aortic root aneurysm and biochemical evidence of increased canonical and noncanonical TGF-ß signaling. Mice that harbor both a mutant Marfan syndrome (MFS) allele (Fbn1(C1039G/+)) and Tgfb2 haploinsufficiency show increased TGF-ß signaling and phenotypic worsening in association with normalization of TGF-ß2 expression and high expression of TGF-ß1. Taken together, these data support the hypothesis that compensatory autocrine and/or paracrine events contribute to the pathogenesis of TGF-ß-mediated vasculopathies.