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.

An unanticipated copy number variant of chromosome 15 disrupting SMAD3 reveals a three-generation family at serious risk for aortic dissection.

Several genes involved in the familial appearance of thoracic aortic aneurysms and dissections (FTAAD) have been characterized recently, one of which is SMAD3. Mutations of SMAD3 cause a new syndromic form of aortic aneurysms and dissections associated with skeletal abnormalities. We discovered a small interstitial deletion of chromosome 15, leading to disruption of SMAD3, in a boy with mild mental retardation, behavioral problems and revealed features of the aneurysms-osteoarthritis syndrome (AOS). Several family members carried the same deletion and showed features including aortic aneurysms and a dissection. This finding demonstrates that haploinsufficiency of SMAD3 leads to development of both thoracic aortic aneurysms and dissections, and the skeletal abnormalities that form part of the aneurysms-osteoarthritis syndrome. Interestingly, the identification of this familial deletion is an example of an unanticipated result of a genomic microarray and led to the discovery of important but unrelated serious aortic disease in the proband and family members.

Ehlers-Danlos arthrochalasia type (VIIA-B)--expanding the phenotype: from prenatal life through adulthood.

The Ehlers-Danlos syndromes (EDS) form a clinically and genetically heterogeneous group of inherited connective-tissue disorders characterized by joint hypermobility, tissue fragility and skin abnormalities. Six subtypes have been well characterized based on clinical features and molecular genetic abnormalities. The arthrochalasia type EDS (formerly types VIIA and B) is characterized by severe generalized joint hypermobility with multiple dislocations including congenital bilateral dislocation of the hips, muscular hypotonia and distinct dysmorphic features. The diagnosis of the arthrochalasia type EDS is of importance in the neonatal period because of consequences of physical disability in later life. However, the differential diagnosis may be difficult because of overlap with other hypermobility syndromes. In addition, the significant hypotonia may direct the physician toward various neuromuscular diagnoses. As patients become older, the hypotonia decreases and facial features become less distinct. In this report, we describe seven patients at different ages. Timing of diagnosis varied from prenatal life to adult age. The diagnosis of EDS type VII was confirmed by biochemical studies or mutation analysis showing characteristic mutations in COL1A1 and COL1A2. These mutations result in skipping of exon 6, which leads to defective collagen synthesis. For physicians treating patients with EDS type VII, achieving mobility for the patient is the greatest challenge and it may be impossible because of recurrent dislocations of nearly all joints in severe cases.

Dilatation of the great arteries in an infant with marfan syndrome and ventricular septal defect.

We describe an infant presenting with contractures of the fingers, a large ventricular septal defect (VSD), and severe pulmonary artery dilatation. He had clinical and echocardiographic features of both neonatal or infantile Marfan syndrome (MFS) and congenital contractural arachnodactyly. After surgical VSD closure, the aortic root developed progressive dilatation while the size of pulmonary artery returned to normal limits. Eventually the diagnosis of MFS was confirmed by DNA analysis.

Recurrent and founder mutations in the Netherlands: Extensive clinical variability in Marfan syndrome patients with a single novel recurrent fibrillin-1 missense mutation.

Background/Methods. Marfan syndrome (MFS) is a heritable connective tissue disorder usually caused by a mutation in the fibrillin 1 (FBN1) gene. Typical characteristics of MFS that have been described include dolichostenomelia, ectopia lentis and aortic root dilatation. However, there is great clinical variability in the expression of the syndrome's manifestations, both between and within families. Here we discuss the clinical variability of MFS by describing a large fourgeneration Dutch family with MFS.Results. Nineteen individuals of one family with a single missense FBN1 mutation (c.7916A>G) were identified. The same mutation was found in one unrelated person. Clinical variability was extensive and not all mutation carriers fulfilled the diagnostic criteria for MFS. Some patients only expressed mild skeletal abnormalities, whereas aortic root dilation was present in eight patients, an acute type A aortic dissection was recorded in two other patients, and a mitral valve prolapse was present in eight patients. In some patients cardiac features were not present on initial screening, but did however develop over time.Conclusion. MFS is a clinically highly variable syndrome, which means a meticulous evaluation of suspected cases is crucial. Mutation carriers should be re-evaluated regularly as cardiovascular symptoms may develop over time. (Neth Heart J 2010;18:85-9.).

Compound-heterozygous Marfan syndrome.

We report two families in which the probands have compound-heterozygous Marfan syndrome (MFS). The proband of family 1 has the R2726W FBN1 mutation associated with isolated skeletal features on one allele and a pathogenic FBN1 mutation on the other allele. The phenotype of the compound-heterozygous probands appears to be more severe than that of their heterozygous family members which underlines the possibility that certain trans-located FBN1 mutations might act as modifiers of phenotype explaining some of the intrafamilial variability in Marfan syndrome.

Variable phenotypes associated with 10q23 microdeletions involving the PTEN and BMPR1A genes.

Infantile juvenile polyposis is a rare disease with severe gastrointestinal symptoms and a grave clinical course. Recently, 10q23 microdeletions involving the PTEN and BMPR1A genes were found in four patients with infantile juvenile polyposis. It was hypothesized that a combined and synergistic effect of the deletion of both genes would explain the condition. Subsequently, however, a patient with a larger 10q23 deletion including the same genes but with a mild clinical phenotype was identified. Here, we present four additional patients with 10q23 microdeletions involving the PTEN and BMPR1A genes. The sizes of the deletions were analyzed using single nucleotide polymorphism array analysis. All patients had macrocephaly, dysmorphic features, retardation and congenital abnormalities. One patient developed colorectal cancer. However, only one case had disease onset before 2 years of age and severe symptoms requiring colectomy. No clear correlation was found between ages at onset or severity of gastrointestinal symptoms and the sizes of the deletions. We conclude that patients with 10q23 microdeletions involving the PTEN and BMPR1A genes have variable clinical phenotypes, which cannot be explained merely by the deletion sizes. The phenotypes are not restricted to severe infantile juvenile polyposis but include childhood-onset cases with macrocephaly, retardation, mild gastrointestinal symptoms and possibly early-onset colorectal cancer.

A 6Mb deletion in band 2q22 due to a complex chromosome rearrangement associated with severe psychomotor retardation, microcephaly and distinctive dysmorphic facial features.

High-resolution analyses of complex chromosome rearrangements (CCR) have demonstrated in individuals with abnormal phenotypes that not all seemingly balanced CCRs based on G-banding are completely balanced at breakpoint level. Here we report on an apparently balanced de novo CCR involving chromosomes 2, 3 and 5 present in a 6-month-old girl. She was referred for genetic evaluation because of severe psychomotor retardation, distinctive dysmorphic features and microcephaly. A 1Mb resolution array-CGH analysis of DNA from the patient revealed a deletion of about 6Mb for chromosome 2. FISH analysis showed that the deletion interval found in band 2q22 mapped at the translocation breakpoint, and that the ZFHX1B gene, which is known to be involved in the Mowat-Wilson syndrome, is located within the deletion interval. To our knowledge this is the first case of a complex chromosomal rearrangement associated with Mowat-Wilson syndrome. Our data illustrate the important role for high-resolution investigation of apparently balanced chromosome rearrangements in patients with unexplained psychomotor retardation and/or other clinical features, and should contribute to our understanding of the mechanisms involved in chromosome rearrangement.

Homozygous and heterozygous expression of a novel insulin-like growth factor-I mutation.

IGF-I is a key factor in intrauterine development and postnatal growth and metabolism. The secretion of IGF-I in utero is not dependent on GH, whereas in childhood and adult life, IGF-I secretion seems to be mainly controlled by GH, as revealed from studies on patients with GHRH receptor and GH receptor mutations. In a 55-yr-old male, the first child of consanguineous parents, presenting with severe intrauterine and postnatal growth retardation, microcephaly, and sensorineural deafness, we found a homozygous G to A nucleotide substitution in the IGF-I gene changing valine 44 into methione. The inactivating nature of the mutation was proven by functional analysis demonstrating a 90-fold reduced affinity of recombinantly produced for the IGF-I receptor. Additional investigations revealed osteoporosis, a partial gonadal dysfunction, and a relatively well-preserved cardiac function. Nine of the 24 relatives studied carried the mutation. They had a significantly lower birth weight, final height, and head circumference than noncarriers. In conclusion, the phenotype of our patient consists of severe intrauterine growth retardation, deafness, and mental retardation, reflecting the GH-independent secretion of IGF-I in utero. The postnatal growth pattern, similar to growth of untreated GH-deficient or GH-insensitive children, is in agreement with the hypothesis that IGF-I secretion in childhood is mainly GH dependent. Remarkably, IGF-I deficiency is relatively well tolerated during the subsequent four decades of adulthood. IGF-I haploinsufficiency results in subtle inhibition of intrauterine and postnatal growth.

Individuals with abnormal phenotype and normal G-banding karyotype: improvement and limitations in the diagnosis by the use of 24-colour FISH.

The simultaneous identification, by fluorescence in situ hybridisation (FISH), of each chromosome in a distinct colour became feasible a few years ago. The key question in the application of this and many other developments in molecular cytogenetics to clinical situations is whether the results add significant further information that is relevant to the diagnosis. So far, limited data exist regarding how much improvement the technique brings to the diagnosis of phenotypically abnormal individuals in whom no abnormalities have been detected by conventional G-banding analysis. Because of the lack of a conclusive diagnosis, genetic counselling, estimation of recurrence risk and prenatal diagnosis of these individuals and their relatives is problematic. We report a study with 24-colour whole-chromosome painting of 10 familial and 11 isolated cases with abnormal phenotypes and normal G-banding karyotypes. Previously undetected unbalanced translocations were revealed in two cases. The value and current cost-effectiveness of multicolour FISH for cytogenetic diagnosis is discussed.

Radial aplasia, poikiloderma and auto-immune enterocolitis--new syndrome or severe form of Rothmund-Thomson syndrome?

A syndrome is described in three isolated patients in whom the main features are bilateral radial aplasia, short stature, an inflammatory based 'elastic' pyloric stenosis, a pan-enteric inflammatory gut disorder that appears to be due to an autoimmune process, and poikiloderma. Other features in individual cases include cleft palate, micrognathia, anal atresia, patellar aplasia/hypoplasia and sensorineural deafness. This combination may represent a severe form of Rothmund-Thomson syndrome or possibly a previously unrecognized condition.

Noninvasive test for fragile X syndrome, using hair root analysis.

Identification of the FMR1 gene and the repeat-amplification mechanism causing fragile X syndrome led to development of reliable DNA-based diagnostic methods, including Southern blot hybridization and PCR. Both methods are performed on DNA isolated from peripheral blood cells and measure the repeat size in FMR1. Using an immunocytochemical technique on blood smears, we recently developed a novel test for identification of patients with fragile X syndrome. This method, also called "antibody test," uses monoclonal antibodies against the FMR1 gene product (FMRP) and is based on absence of FMRP in patients' cells. Here we describe a new diagnostic test to identify male patients with fragile X syndrome, on the basis of lack of FMRP in their hair roots. Expression of FMRP in hair roots was studied by use of an FMRP-specific antibody test, and the percentage of FMRP-expressing hair roots in controls and in male fragile X patients was determined. Control individuals showed clear expression of FMRP in nearly every hair root, whereas male fragile X patients lacked expression of FMRP in almost all their hair roots. Mentally retarded female patients with a full mutation showed FMRP expression in only some of their hair roots (<55%), and no overlap with normal female controls was observed. The advantages of this test are (1) plucking of hair follicles does no appreciable harm to the mentally retarded patient, (2) hairs can be sent in a simple envelope to a diagnostic center, and (3) the result of the test is available within 5 h of plucking. In addition, this test enabled us to identify two fragile X patients who did not show the full mutation by analysis of DNA isolated from blood cells.

The autosomal dominant syndrome with congenital stapes ankylosis, broad thumbs and hyperopia.

A family is reported with conductive hearing loss, hyperopia, broad thumbs and broad first toes. The family resembles a previous reported family (Teunissen B, Cremers CWRJ (1990) Laryngoscope 100: 380-384) but additionally all affected members have a typical face. Overlap of the Teunissen and Cremers syndrome with the facio-audio-symphalangism syndrome and proximal symphalangism is discussed.