Nizar Smaoui

Clinical application of whole-exome sequencing across clinical indications

Purpose:We report the diagnostic yield of whole-exome sequencing (WES) in 3,040 consecutive cases at a single clinical laboratory.Methods:WES was performed for many different clinical indications and included the proband plus two or more family members in 76% of cases.Results:The overall diagnostic yield of WES was 28.8%. The diagnostic yield was 23.6% in proband-only cases and 31.0% when three family members were analyzed. The highest yield was for patients who had disorders involving hearing (55%, N = 11), vision (47%, N = 60), the skeletal muscle system (40%, N = 43), the skeletal system (39%, N = 54), multiple congenital anomalies (36%, N = 729), skin (32%, N = 31), the central nervous system (31%, N = 1,082), and the cardiovascular system (28%, N = 54). Of 2,091 cases in which secondary findings were analyzed for 56 American College of Medical Genetics and Genomics–recommended genes, 6.2% (N = 129) had reportable pathogenic variants. In addition to cases with a definitive diagnosis, in 24.2% of cases a candidate gene was reported that may later be reclassified as being associated with a definitive diagnosis.Conclusion:Our experience with our first 3,040 WES cases suggests that analysis of trios significantly improves the diagnostic yield compared with proband-only testing for genetically heterogeneous disorders and facilitates identification of novel candidate genes.Genet Med 18 7, 696–704.

Exon-level array CGH in a large clinical cohort demonstrates increased sensitivity of diagnostic testing for Mendelian disorders

Purpose:Mendelian disorders are most commonly caused by mutations identifiable by DNA sequencing. Exonic deletions and duplications can go undetected by sequencing, and their frequency in most Mendelian disorders is unknown.Methods:We designed an array comparative genomic hybridization (CGH) test with probes in exonic regions of 589 genes. Targeted testing was performed for 219 genes in 3,018 patients. We demonstrate for the first time the utility of exon-level array CGH in a large clinical cohort by testing for 136 autosomal dominant, 53 autosomal recessive, and 30 X-linked disorders.Results:Overall, 98 deletions and two duplications were identified in 53 genes, corresponding to a detection rate of 3.3%. Approximately 40% of positive findings were deletions of only one or two exons. A high frequency of deletions was observed for several autosomal dominant disorders, with a detection rate of 2.9%. For autosomal recessive disorders, array CGH was usually performed after a single mutation was identified by sequencing. Among 138 individuals tested for recessive disorders, 10.1% had intragenic deletions. For X-linked disorders, 3.5% of 313 patients carried a deletion or duplication.Conclusion:Our results demonstrate that exon-level array CGH provides a robust option for intragenic copy number analysis and should routinely supplement sequence analysis for Mendelian disorders.Genet Med 2012:14(6):594–603

De novo mutations in KIF1A cause progressive encephalopathy and brain atrophy

To determine the cause and course of a novel syndrome with progressive encephalopathy and brain atrophy in children.

Abnormal Cone Structure in Foveal Schisis Cavities in X-Linked Retinoschisis from Mutations in Exon 6 of the RS1 Gene

PURPOSE To evaluate macular cone structure in patients with X-linked retinoschisis (XLRS) caused by mutations in exon 6 of the RS1 gene. METHODS High-resolution macular images were obtained with adaptive optics scanning laser ophthalmoscopy (AOSLO) and spectral domain optical coherence tomography (SD-OCT) in two patients with XLRS and 27 age-similar healthy subjects. Retinal structure was correlated with best-corrected visual acuity, kinetic and static perimetry, fundus-guided microperimetry, full-field electroretinography (ERG), and multifocal ERG. The six coding exons and the flanking intronic regions of the RS1 gene were sequenced in each patient. RESULTS Two unrelated males, ages 14 and 29, with visual acuity ranging from 20/32 to 20/63, had macular schisis with small relative central scotomas in each eye. The mixed scotopic ERG b-wave was reduced more than the a-wave. SD-OCT showed schisis cavities in the outer and inner nuclear and plexiform layers. Cone spacing was increased within the largest foveal schisis cavities but was normal elsewhere. In each patient, a mutation in exon 6 of the RS1 gene was identified and was predicted to change the amino acid sequence in the discoidin domain of the retinoschisin protein. CONCLUSIONS AOSLO images of two patients with molecularly characterized XLRS revealed increased cone spacing and abnormal packing in the macula of each patient, but cone coverage and function were near normal outside the central foveal schisis cavities. Although cone density is reduced, the preservation of wave-guiding cones at the fovea and eccentric macular regions has prognostic and therapeutic implications for XLRS patients with foveal schisis. (Clinical Trials.gov number, NCT00254605.).

Advanced bone age in a girl with Wiedemann–Steiner syndrome and an exonic deletion in KMT2A (MLL)

Recognition of the gene implicated in a Mendelian disorder subsequently leads to an expansion of potential phenotypes associated with mutations in that gene as patients with features beyond the core phenotype are identified by sequencing. Here, we present a young girl with developmental delay, short stature despite a markedly advanced bone age, hypertrichosis without elbow hair, renal anomalies, and dysmorphic facial features, found to have a heterozygous, de novo, intragenic deletion encompassing exons 2–10 of the KMT2A (MLL) gene detected by whole exome sequencing. Heterozygous mutations in this gene were recently demonstrated to cause Wiedemann–Steiner syndrome (OMIM 605130). Importantly, retrospective analysis of this patients chromosomal microarray revealed decreased copy number of two probes corresponding to exons 2 and 9 of the KMT2A gene, though this result was not reported by the testing laboratory in keeping with standard protocols for reportable size cutoffs for array comparative genomic hybridization. This patient expands the clinical phenotype associated with mutations in KMT2A to include variable patterns of hypertrichosis and a significantly advanced bone age with premature eruption of the secondary dentition despite her growth retardation. This patient also represents the first report of Wiedemann–Steiner syndrome due to an exonic deletion, supporting haploinsufficiency as a causative mechanism. Our patient also illustrates the need for sensitive guidelines for the reporting of chromosomal microarray findings that are below traditional reporting size cutoffs, but that impact exons or other genomic regions of known function.

DNA variant databases improve test accuracy and phenotype prediction in Alport syndrome.

X-linked Alport syndrome is a form of progressive renal failure caused by pathogenic variants in the COL4A5 gene. More than 700 variants have been described and a further 400 are estimated to be known to individual laboratories but are unpublished. The major genetic testing laboratories for X-linked Alport syndrome worldwide have established a Web-based database for published and unpublished COL4A5 variants (https://grenada.lumc.nl/LOVD2/COL4A/home.php?select_db=COL4A5). This conforms with the recommendations of the Human Variome Project: it uses the Leiden Open Variation Database (LOVD) format, describes variants according to the human reference sequence with standardized nomenclature, indicates likely pathogenicity and associated clinical features, and credits the submitting laboratory. The database includes non-pathogenic and recurrent variants, and is linked to another COL4A5 mutation database and relevant bioinformatics sites. Access is free. Increasing the number of COL4A5 variants in the public domain helps patients, diagnostic laboratories, clinicians, and researchers. The database improves the accuracy and efficiency of genetic testing because its variants are already categorized for pathogenicity. The description of further COL4A5 variants and clinical associations will improve our ability to predict phenotype and our understanding of collagen IV biochemistry. The database for X-linked Alport syndrome represents a model for databases in other inherited renal diseases.

Genomics in the era of molecular ophthalmology: reflections on the National Ophthalmic Disease Genotyping Network (eyeGENE).

That the Archives of Ophthalmology is devoting this issue to genomics in ophthalmology just over one year from its two issues on genetics in ophthalmology is a testament to that fact that the era of genomic medicine is rapidly being incorporated into ophthalmology.(1) The evolution of genetic medicine has been accelerated following the full sequencing of the human genome,(2) the HapMap Project,(3) and the identification important genetic components to complex diseases such as macular degeneration and glaucoma.(4–7) Science has moved forward at a rapid pace. Now is the time to put this knowledge into clinical practice. Approximately 71% of respondents to a recent survey wanted more information from their physicians about genetic conditions in their family.(8) Our patients are looking for us as ophthalmologists to take the lead.

Novel de novo SPOCK1 mutation in a proband with developmental delay, microcephaly and agenesis of corpus callosum.

Whole exome sequencing made it possible to identify novel de novo mutations in genes that might be linked to human syndromes (genotype first analysis). We describe a female patient with a novel de novo SPOCK1 variant, which has not been previously been associated with a human phenotype. Her features include intellectual disability with dyspraxia, dysarthria, partial agenesis of corpus callosum, prenatal-onset microcephaly and atrial septal defect with aberrant subclavian artery. Previous genetic, cytogenomic and metabolic studies were unrevealing. At age 13 years, exome sequencing on the patient and her parents revealed a de novo novel missense mutation in SPOCK1 (coding for Testican-1) on chromosome 5q31: c.239A>T (p.D80V). This mutation affects a highly evolutionarily conserved area of the gene, replacing a polar aspartic acid with hydrophobic nonpolar valine, and changing the chemical properties of the protein product, likely representing a pathogenic variant. Previous microdeletions of 5q31 including SPOCK1 have suggested genes on 5q31 as candidates for intellectual disability. No mutations or variants in other genes potentially linked to her phenotype were identified. Testicans are proteoglycans belonging to the BM-40/SPARC/osteonectin family of extracellular calcium-binding proteins. Testican-1 is encoded by the SPOCK1 gene, and mouse models have been shown it to be strongly expressed in the brain and to be involved in neurogenesis. We hypothesize that because this gene function is critical for neurogenesis, mutations could potentially lead to a phenotype with developmental delay and microcephaly.

Peripheral retinal nonperfusion in septo-optic dysplasia (de Morsier syndrome)

tocoagulation was applied and the worm was successfully killed (Figure 1F). The patient reported significant subjective improvement, and his visual acuity measured 20/40 OS 1 month and 20/25 OS 3 months following the initial visit. Spectraldomain OCT showed disruption of the photoreceptors in the papillomacular region secondary to prior photocoagulation but progressive restoration of the normal inner segment/outer segment junction and photoreceptor architecture in the fovea (Figure 2B and C).

De novo duplication 11p13 involving the PAX6 gene in a patient with neonatal seizures, hypotonia, microcephaly, developmental disability and minor ocular manifestations

De Novo Duplication 11p13 Involving the PAX6 Gene in a Patient with Neonatal Seizures, Hypotonia, Microcephaly, Developmental Disability and Minor Ocular Manifestations Swaroop Aradhya,* Nizar Smaoui, Michael Marble, and Yves Lacassie** GeneDx, Gaithersburg, Maryland Department of Pediatrics, Louisiana State University Health Sciences Center, New Orleans, and Children’s Hospital, New Orleans, Louisiana