NGS panel analysis in 24 ectopia lentis patients; a clinically relevant test with a high diagnostic yield.

BACKGROUND: Several genetic causes of ectopia lentis (EL), with or without systemic features, are known. The differentiation between syndromic and isolated EL is crucial for further treatment, surveillance and counseling of patients and their relatives. Next generation sequencing (NGS) is a powerful tool enabling the simultaneous, highly-sensitive analysis of multiple target genes. OBJECTIVE: The aim of this study was to evaluate the diagnostic yield of our NGS panel in EL patients. Furthermore, we provide an overview of currently described mutations in ADAMTSL4, the main gene involved in isolated EL. METHODS: A NGS gene panel was analysed in 24 patients with EL. RESULTS: A genetic diagnosis was confirmed in 16 patients (67%). Of these, four (25%) had a heterozygous FBN1 mutation, 12 (75%) were homozygous or compound heterozygous for ADAMTSL4 mutations. The known European ADAMTSL4 founder mutation c.767_786del was most frequently detected. CONCLUSION: The diagnostic yield of our NGS panel was high. Causative mutations were exclusively identified in ADAMTSL4 and FBN1. With this approach the risk of misdiagnosis or delayed diagnosis can be reduced. The value and clinical implications of establishing a genetic diagnosis in patients with EL is corroborated by the description of two patients with an unexpected underlying genetic condition.

After the introduction into the national newborn screening program: who is receiving genetic counseling for hemoglobinopathies in the Netherlands?

OBJECTIVE: Universal newborn screening for hemoglobinopathies started in The Netherlands in 2007. Herewith severe conditions, such as sickle cell disease, ß-thalassemia major and hemoglobin H disease are putatively identified. Additionally, at least 1,800 carriers of hemoglobin variants associated with severe conditions in homozygote or compound heterozygote forms are identified yearly. Thus far, approximately 60 patients and 800 healthy sickle cell (HbS) carriers are reported each year among 180,000 newborns. Results are sent to the general practitioner with the recommendation to inform and diagnose both parents of the healthy carriers to exclude genetic risk, while patients and their parents are referred directly to a pediatrician. This study was performed to determine how often parents of identified carriers and affected newborns are seen in genetic centers for counseling. METHODS: In this retrospective study, we collected anonymized data from 7 of the 8 Dutch clinical genetic centers from January 1, 2007, until December 31, 2010. RESULTS: After an initial general increase in total counseling intakes, a decline was noticed in the third year, while the requests for prenatal diagnoses remained relatively stable. In 2007 and 2013, genetic counselors were asked for self-reported knowledge. They found hemoglobinopathy counseling complex, but by 2013, they indicated they had acquired sufficient knowledge on most hemoglobinopathy aspects. CONCLUSION: We could not observe a significant increase in genetic counseling for hemoglobinopathy after its introduction into newborn screening. Although 120 HbS carriers and 60 patients are expected to be born from couples at risk annually, only 33 at risk couples out of 540 families of diagnosed newborns received optimal care and information at a genetics center in 4 years.

Cytoplasmic localization of PML particles in laminopathies.

There is growing evidence that laminopathies, diseases associated with mutations in the LMNA gene, are caused by a combination of mechanical and gene regulatory distortions. Strikingly, there is a large variability in disease symptoms between individual patients carrying an identical LMNA mutation. This is why classical genetic screens for mutations appear to have limited predictive value for disease development. Recently, the widespread occurrence of repetitive nuclear ruptures has been described in fibroblast cultures from various laminopathy patients. Since this phenomenon was strongly correlated with disease severity, the identification of biomarkers that report on these rupture events could have diagnostic relevance. One such candidate marker is the PML nuclear body, a structure that is normally confined to the nuclear interior, but leaks out of the nucleus upon nuclear rupture. Here, we show that a variety of laminopathies shows the presence of these cytoplasmic PML particles (PML CPs), and that the amount of these protein aggregates increases with severity of the disease. In addition, between clinically healthy individuals, carrying LMNA mutations, significant differences can be found. Therefore, we postulate that detection of PML CPs in patient fibroblasts could become a valuable marker for diagnosis of disease development.

Congenital hydrocephalus in clinical practice: a genetic diagnostic approach.

Congenital hydrocephalus is a common and often disabling disorder. The etiology is very heterogeneous. Little is known about the genetic causes of congenital hydrocephalus. A retrospective survey was performed including patients with primary congenital hydrocephalus referred to the Department of Clinical Genetics between 1985 and 2010 by perinatologists, (child) neurologists or pediatricians. Patients with hydrocephalus secondary to other pathology were excluded from this survey. We classified patients with primary congenital hydrocephalus into two main groups: non-syndromic hydrocephalus (NSH) and syndromic hydrocephalus (SH). Seventy-five individuals met the inclusion criteria, comprising 36% (27/75) NSH and 64% (48/75) SH. In 11% (8/75) hydrocephalus was familial. The cause of hydrocephalus was unknown in 81% (61/75), including all patients with NSH. The male-female ratio in this subgroup was 2.6:1, indicating an X-linked factor other than the L1CAM gene. In the group of SH patients, 29% (14/48) had a known cause of hydrocephalus including chromosomal abnormalities, L1 syndrome, Marden-Walker syndrome, Walker-Warburg syndrome and hemifacial microsomia. We performed this survey in order to evaluate current knowledge on the genetic etiology of primary congenital hydrocephalus and to identify new candidate genes or regulatory pathways for congenital hydrocephalus. Recommendations were made concerning the evaluation and genetic workup of patients with primary congenital hydrocephalus. We conclude that further molecular and functional analysis is needed to identify new genetic forms of congenital hydrocephalus.

Hereditary leiomyomatosis and renal cell cancer in families referred for fumarate hydratase germline mutation analysis.

Heterozygous fumarate hydratase (FH) germline mutations cause hereditary leiomyomatosis and renal cell cancer (HLRCC), an autosomal dominant syndrome characterized by multiple cutaneous piloleiomyomas, uterine leiomyomas and papillary type 2 renal cancer. The main objective of our study was to evaluate clinical and genetic data from families suspected of HLRCC on a nationwide level. All families referred for FH mutation analysis in the Netherlands were assessed. We performed FH sequence analysis and multiplex ligation-dependent probe amplification. Families with similar FH mutations were examined for haplotype sharing. In 14 out of 33 families, we identified 11 different pathogenic FH germline mutations, including 4 novel mutations and 1 whole-gene deletion. Clinical data were available for 35 FH mutation carriers. Cutaneous leiomyomas were present in all FH mutation carriers older than 40 years of age. Eleven out of 21 female FH mutation carriers underwent surgical treatment for symptomatic uterine leiomyomas at an average of 35 years. Two FH mutation carriers had papillary type 2 renal cancer and Wilms' tumour, respectively. We evaluated the relevance of our findings for clinical practice and have proposed clinical diagnostic criteria, indications for FH mutation analysis and recommendations for management.

Founder mutations in the Netherlands: SCN5a 1795insD, the first described arrhythmia overlap syndrome and one of the largest and best characterised families worldwide.

In this part of a series on founder mutations in the Netherlands, we review a Dutch family carrying the SCN5a 1795insD mutation. We describe the advances in our understanding of the premature sudden cardiac deaths that have accompanied this family in the past centuries. The mutation carriers show a unique overlap of long-QT syndrome (type 3), Brugada syndrome and progressive cardiac conduction defects attributed to a single mutation in the cardiac sodium channel gene SCN5a. It is at present one of the largest and best-described families worldwide and we have learned immensely from the mouse strains with the murine homologue of the SCN5a 1795insD mutation (SCN5a 1798insD). From the studies currently performed we are about to obtain new insights into the phenotypic variability in this monogenic arrhythmia syndrome, and this might also be relevant for other arrhythmia syndromes and the general population. (Neth Heart J 2009;17:422-8.).

Cleft palate cells can regenerate a palatal mucosa in vitro.

Cleft palate repair leaves full-thickness mucosal defects on the palate. Healing might be improved by implantation of a mucosal substitute. However, the genetic and phenotypic deviations of cleft palate cells may hamper tissue engineering. The aim of this study was to construct mucosal substitutes from cleft palate cells, and to compare these with substitutes from normal palatal cells, and with native palatal mucosa. Biopsies from the palatal mucosa of eight children with cleft palate and eight age-matched control individuals were taken. Three biopsies of both groups were processed for (immuno)histochemistry; 5 were used to culture mucosal substitutes. Histology showed that the substitutes from cleft-palate and non-cleft-palate cells were comparable, but the number of cell layers was less than in native palatal mucosa. All epithelial layers in native palatal mucosa and mucosal substitutes expressed the cytokeratins 5, 10, and 16, and the proliferation marker Ki67. Heparan sulphate and decorin were present in the basal membrane and the underlying connective tissue, respectively. We conclude that mucosal cells from children with cleft palate can regenerate an oral mucosa in vitro.