Roberto Mendoza-Londono

Characterization of Potocki-Lupski Syndrome (dup(17)(p11.2p11.2)) and Delineation of a Dosage-Sensitive Critical Interval That Can Convey an Autism Phenotype

The duplication 17p11.2 syndrome, associated with dup(17)(p11.2p11.2), is a recently recognized syndrome of multiple congenital anomalies and mental retardation and is the first predicted reciprocal microduplication syndrome described--the homologous recombination reciprocal of the Smith-Magenis syndrome (SMS) microdeletion (del(17)(p11.2p11.2)). We previously described seven subjects with dup(17)(p11.2p11.2) and noted their relatively mild phenotype compared with that of individuals with SMS. Here, we molecularly analyzed 28 additional patients, using multiple independent assays, and also report the phenotypic characteristics obtained from extensive multidisciplinary clinical study of a subset of these patients. Whereas the majority of subjects (22 of 35) harbor the homologous recombination reciprocal product of the common SMS microdeletion (~3.7 Mb), 13 subjects (~37%) have nonrecurrent duplications ranging in size from 1.3 to 15.2 Mb. Molecular studies suggest potential mechanistic differences between nonrecurrent duplications and nonrecurrent genomic deletions. Clinical features observed in patients with the common dup(17)(p11.2p11.2) are distinct from those seen with SMS and include infantile hypotonia, failure to thrive, mental retardation, autistic features, sleep apnea, and structural cardiovascular anomalies. We narrow the critical region to a 1.3-Mb genomic interval that contains the dosage-sensitive RAI1 gene. Our results refine the critical region for Potocki-Lupski syndrome, provide information to assist in clinical diagnosis and management, and lend further support for the concept that genomic architecture incites genomic instability.

De novo ACTA2 mutation causes a novel syndrome of multisystemic smooth muscle dysfunction

Smooth muscle cells (SMCs) contract to perform many physiological functions, including regulation of blood flow and pressure in arteries, contraction of the pupils, peristalsis of the gut, and voiding of the bladder. SMC lineage in these organs is characterized by cellular expression of the SMC isoform of α‐actin, encoded by the ACTA2 gene. We report here on a unique and de novo mutation in ACTA2, R179H, that causes a syndrome characterized by dysfunction of SMCs throughout the body, leading to aortic and cerebrovascular disease, fixed dilated pupils, hypotonic bladder, malrotation, and hypoperistalsis of the gut and pulmonary hypertension.

Utility of whole‐exome sequencing for those near the end of the diagnostic odyssey: time to address gaps in care

An accurate diagnosis is an integral component of patient care for children with rare genetic disease. Recent advances in sequencing, in particular whole‐exome sequencing (WES), are identifying the genetic basis of disease for 25–40% of patients. The diagnostic rate is probably influenced by when in the diagnostic process WES is used. The Finding Of Rare Disease GEnes (FORGE) Canada project was a nation‐wide effort to identify mutations for childhood‐onset disorders using WES. Most children enrolled in the FORGE project were toward the end of the diagnostic odyssey. The two primary outcomes of FORGE were novel gene discovery and the identification of mutations in genes known to cause disease. In the latter instance, WES identified mutations in known disease genes for 105 of 362 families studied (29%), thereby informing the impact of WES in the setting of the diagnostic odyssey. Our analysis of this dataset showed that these known disease genes were not identified prior to WES enrollment for two key reasons: genetic heterogeneity associated with a clinical diagnosis and atypical presentation of known, clinically recognized diseases. What is becoming increasingly clear is that WES will be paradigm altering for patients and families with rare genetic diseases.

Mutations in PLK4, encoding a master regulator of centriole biogenesis, cause microcephaly, growth failure and retinopathy

Centrioles are essential for ciliogenesis. However, mutations in centriole biogenesis genes have been reported in primary microcephaly and Seckel syndrome, disorders without the hallmark clinical features of ciliopathies. Here we identify mutations in the genes encoding PLK4 kinase, a master regulator of centriole duplication, and its substrate TUBGCP6 in individuals with microcephalic primordial dwarfism and additional congenital anomalies, including retinopathy, thereby extending the human phenotypic spectrum associated with centriole dysfunction. Furthermore, we establish that different levels of impaired PLK4 activity result in growth and cilia phenotypes, providing a mechanism by which microcephaly disorders can occur with or without ciliopathic features.

The clinical application of genome-wide sequencing for monogenic diseases in Canada: Position Statement of the Canadian College of Medical Geneticists

Purpose and scope The aim of this Position Statement is to provide recommendations for Canadian medical geneticists, clinical laboratory geneticists, genetic counsellors and other physicians regarding the use of genome-wide sequencing of germline DNA in the context of clinical genetic diagnosis. This statement has been developed to facilitate the clinical translation and development of best practices for clinical genome-wide sequencing for genetic diagnosis of monogenic diseases in Canada; it does not address the clinical application of this technology in other fields such as molecular investigation of cancer or for population screening of healthy individuals. Methods of statement development Two multidisciplinary groups consisting of medical geneticists, clinical laboratory geneticists, genetic counsellors, ethicists, lawyers and genetic researchers were assembled to review existing literature and guidelines on genome-wide sequencing for clinical genetic diagnosis in the context of monogenic diseases, and to make recommendations relevant to the Canadian context. The statement was circulated for comment to the Canadian College of Medical Geneticists (CCMG) membership-at-large and, following incorporation of feedback, approved by the CCMG Board of Directors. The CCMG is a Canadian organisation responsible for certifying medical geneticists and clinical laboratory geneticists, and for establishing professional and ethical standards for clinical genetics services in Canada. Results and conclusions Recommendations include (1) clinical genome-wide sequencing is an appropriate approach in the diagnostic assessment of a patient for whom there is suspicion of a significant monogenic disease that is associated with a high degree of genetic heterogeneity, or where specific genetic tests have failed to provide a diagnosis; (2) until the benefits of reporting incidental findings are established, we do not endorse the intentional clinical analysis of disease-associated genes other than those linked to the primary indication; and (3) clinicians should provide genetic counselling and obtain informed consent prior to undertaking clinical genome-wide sequencing. Counselling should include discussion of the limitations of testing, likelihood and implications of diagnosis and incidental findings, and the potential need for further analysis to facilitate clinical interpretation, including studies performed in a research setting. These recommendations will be routinely re-evaluated as knowledge of diagnostic and clinical utility of clinical genome-wide sequencing improves. While the document was developed to direct practice in Canada, the applicability of the statement is broader and will be of interest to clinicians and health jurisdictions internationally.

Generalized metabolic bone disease in Neurofibromatosis type I

Skeletal abnormalities are a recognized component of Neurofibromatosis type I (NF1) but a generalized metabolic bone defect in NF1 has not been fully characterized thus far. The purpose of this study was to characterize at the densitometric, biochemical and pathological level the bone involvement in NF1 patients. Using dual energy X-ray absorptiometry (DXA) we analyzed bone status in 73 unselected NF1 subjects, 26 males and 47 females, mainly children and adolescents (mean age: 16.6 years). In a subgroup of subjects with low bone mass, we measured indices of calcium-phosphate metabolism, bone turnover, and bone density before and after vitamin D and calcium treatment. We found statistically significant and generalized reduction in bone mass with the mean lumbar bone mineral density (BMD) z-score being -1.38+/-1.05 (CI 95% -1.62 to -1.13), and whole body bone mineral content (BMC) z-score -0.61+/-1.19 (CI 95% -0.94 to -0.29), both significantly reduced compared to normal controls (p<.001). PTH was moderately elevated and after 4 months of supplemental therapy with calcium and vitamin D, it decreased to the normal range. However, BMD z-scores did not significantly improve after 2 years of follow-up. Histological analysis of bone samples from NF1 patients revealed substantial alteration of bone microarchitecture due mainly to reduced trabecular bone. Our observations are consistent with a generalized bone metabolic defect due to loss of the function of neurofibromin. Early identification of patients with osteoporosis may permit more timely and aggressive treatments to prevent the likely substantial morbidity associated with increased fracture risk later in life.

Spondylo-epiphyseal dysplasia, Maroteaux type (pseudo-Morquio syndrome type 2),and parastremmatic dysplasia are caused by TRPV4 mutations.

Recent discoveries have established the existence of a family of skeletal dysplasias caused by dominant mutations in TRPV4. This family comprises, in order of increasing severity, dominant brachyolmia, spondylo‐metaphyseal dysplasia Kozlowski type, and metatropic dysplasia. We tested the hypothesis that a further condition, Spondylo‐epiphyseal dysplasia (SED), Maroteaux type (MIM 184095; also known as pseudo‐Morquio syndrome type 2), could be caused by TRPV4 mutations. We analyzed six individuals with Maroteaux type SED, including three who had previously been reported. All six patients were found to have heterozygous TRPV4 mutations; three patients had unreported mutations, while three patients had mutations previously described in association with metatropic dysplasia. In addition, we tested one individual with a distinct rare disorder, parastremmatic dysplasia (MIM 168400). This patient had a common, recurrent mutation seen in several patients with Kozlowski type spondylo‐metaphyseal dysplasia. We conclude that SED Maroteaux type and parastremmatic dysplasia are part of the TRPV4 dysplasia family and that TRPV4 mutations show considerable variability in phenotypic expression resulting in distinct clinical‐radiographic phenotypes.

Mutations in B3GALT6, which Encodes a Glycosaminoglycan Linker Region Enzyme, Cause a Spectrum of Skeletal and Connective Tissue Disorders

Proteoglycans (PGs) are a major component of the extracellular matrix in many tissues and function as structural and regulatory molecules. PGs are composed of core proteins and glycosaminoglycan (GAG) side chains. The biosynthesis of GAGs starts with the linker region that consists of four sugar residues and is followed by repeating disaccharide units. By exome sequencing, we found that B3GALT6 encoding an enzyme involved in the biosynthesis of the GAG linker region is responsible for a severe skeletal dysplasia, spondyloepimetaphyseal dysplasia with joint laxity type 1 (SEMD-JL1). B3GALT6 loss-of-function mutations were found in individuals with SEMD-JL1 from seven families. In a subsequent candidate gene study based on the phenotypic similarity, we found that B3GALT6 is also responsible for a connective tissue disease, Ehlers-Danlos syndrome (progeroid form). Recessive loss-of-function mutations in B3GALT6 result in a spectrum of disorders affecting a broad range of skeletal and connective tissues characterized by lax skin, muscle hypotonia, joint dislocation, and spinal deformity. The pleiotropic phenotypes of the disorders indicate that B3GALT6 plays a critical role in a wide range of biological processes in various tissues, including skin, bone, cartilage, tendon, and ligament.

Recessive Osteogenesis Imperfecta Caused by Missense Mutations in SPARC

Secreted protein, acidic, cysteine-rich (SPARC) is a glycoprotein that binds to collagen type I and other proteins in the extracellular matrix. Using whole-exome sequencing to identify the molecular defect in two unrelated girls with severe bone fragility and a clinical diagnosis of osteogenesis imperfecta type IV, we identified two homozygous variants in SPARC (GenBank: NM_003118.3; c.497G>A [p.Arg166His] in individual 1; c.787G>A [p.Glu263Lys] in individual 2). Published modeling and site-directed mutagenesis studies had previously shown that the residues substituted by these mutations form an intramolecular salt bridge in SPARC and are essential for the binding of SPARC to collagen type I. The amount of SPARC secreted by skin fibroblasts was reduced in individual 1 but appeared normal in individual 2. The migration of collagen type I alpha chains produced by these fibroblasts was mildly delayed on SDS-PAGE gel, suggesting some overmodification of collagen during triple helical formation. Pulse-chase experiments showed that collagen type I secretion was mildly delayed in skin fibroblasts from both individuals. Analysis of an iliac bone sample from individual 2 showed that trabecular bone was hypermineralized on the material level. In conclusion, these observations show that homozygous mutations in SPARC can give rise to severe bone fragility in humans.

Molecular diagnosis of 22q11.2 deletion and duplication by multiplex ligation dependent probe amplification

22q11 Deletion syndrome (22q11DS) is the most common microdeletion syndrome in humans, occurring with an incidence of 1 in 4,000. In most cases the submicroscopic deletion spans 3 Mb, but there are a number of other overlapping and non‐overlapping deletions that generate a similar phenotype. The majority of the 22q11.2 microdeletions can be ascertained using a standard fluorescence in situ hybridization (FISH) assay probing for TUPLE1 or N25 on 22q11.2. However, this test fails to detect deletions that are either proximal or distal to the FISH probes, and does not provide any information about the length of the deletion. In order to increase the detection rate of 22q11.2 deletion and to better characterize the size and position of such deletions we undertook a study of 22q11.2 cases using multiplex ligation dependent probe amplification (MLPA). We used MLPA to estimate the size of the 22q11.2 deletions in 51 patients positive for TUPLE1 or N25 (FISH) testing, and to investigate 12 patients with clinical features suggestive of 22q11DS and negative FISH results. MLPA analysis confirmed a microdeletion in all 51 FISH‐positive samples as well as microduplications in three samples. Further, it allowed us to delineate deletions not previously detected using standard clinical FISH probes in 2 of 12 subjects with clinical features suggestive of 22q11DS. We conclude that MLPA is a cost‐effective and accurate diagnostic tool for 22q11DS with a higher sensitivity than FISH alone. Additional advantages of MLPA testing in our study included determination of deletion length and detection of 22q11.2 duplications.