Robert Conway

De novo germline and postzygotic mutations in AKT3, PIK3R2 and PIK3CA cause a spectrum of related megalencephaly syndromes

Megalencephaly-capillary malformation (MCAP) and megalencephaly-polymicrogyria-polydactyly-hydrocephalus (MPPH) syndromes are sporadic overgrowth disorders associated with markedly enlarged brain size and other recognizable features. We performed exome sequencing in 3 families with MCAP or MPPH, and our initial observations were confirmed in exomes from 7 individuals with MCAP and 174 control individuals, as well as in 40 additional subjects with megalencephaly, using a combination of Sanger sequencing, restriction enzyme assays and targeted deep sequencing. We identified de novo germline or postzygotic mutations in three core components of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway. These include 2 mutations in AKT3, 1 recurrent mutation in PIK3R2 in 11 unrelated families with MPPH and 15 mostly postzygotic mutations in PIK3CA in 23 individuals with MCAP and 1 with MPPH. Our data highlight the central role of PI3K-AKT signaling in vascular, limb and brain development and emphasize the power of massively parallel sequencing in a challenging context of phenotypic and genetic heterogeneity combined with postzygotic mosaicism.

Discovery of a previously unrecognized microdeletion syndrome of 16p11.2–p12.2

We have identified a recurrent de novo pericentromeric deletion in 16p11.2–p12.2 in four individuals with developmental disabilities by microarray-based comparative genomic hybridization analysis. The identification of common clinical features in these four individuals along with the characterization of complex segmental duplications flanking the deletion regions suggests that nonallelic homologous recombination mediated these rearrangements and that deletions in 16p11.2–p12.2 constitute a previously undescribed syndrome.

Megalencephaly‐capillary malformation (MCAP) and megalencephaly‐polydactyly‐polymicrogyria‐hydrocephalus (MPPH) syndromes: Two closely related disorders of brain overgrowth and abnormal brain and body morphogenesis

The macrocephaly‐capillary malformation syndrome (M‐CM), which we here propose to rename the megalencephaly‐capillary malformation syndrome (MCAP; alternatively the megalencephaly‐capillary malformation‐polymicrogyria syndrome), and the more recently described megalencephaly‐polymicrogyria‐polydactyly‐hydrocephalus syndrome (MPPH) are two megalencephaly (MEG) disorders that involve a unique constellation of physical and neuroimaging anomalies. We compare the features in 42 patients evaluated for physical and neuroimaging characteristics of MCAP and MPPH and propose a more global view of these syndromes based on classes of developmental abnormalities that include primary MEG and growth dysregulation, developmental vascular anomalies (primarily capillary malformations), distal limb anomalies (such as syndactyly and polydactyly), cortical brain malformations (most distinctively polymicrogyria, PMG), and variable connective tissue dysplasia. Based on these classes of developmental abnormalities, we propose that MCAP diagnostic criteria include progressive MEG with either vascular anomalies or syndactyly. In parallel, we propose that MPPH diagnostic criteria include progressive MEG and PMG, absence of the vascular anomalies and syndactyly characteristic of MCAP, and absence of brain heterotopia.

Neuroimaging findings in macrocephaly–capillary malformation: A longitudinal study of 17 patients

Here, we report the neuroimaging findings and neurological changes in 17 unpublished patients with Macrocephaly–Capillary Malformation (M–CM). This syndrome has been traditionally known as Macrocephaly–Cutis Marmorata Telangiectatica Congenita (M–CMTC), but we explain why M–CM is a more accurate term for this overgrowth syndrome. We analyzed the 17 patients with available brain MRI or CT scans and compared their findings with features identified by a comprehensive review of published cases. White matter irregularities with increased signal on T2‐weighted images were commonly observed findings. A distinctive feature in more than half the patients was cerebellar tonsillar herniation associated with rapid brain growth and progressive crowding of the posterior fossa during infancy. In four such cases, we confirmed that the tonsillar herniation was an acquired event. Concurrently, with the development of these findings, ventriculomegaly (frequently obstructive) and dilated dural venous sinuses were observed in conjunction with prominent Virchow–Robin spaces in many of those in whom cerebellar tonsil herniation had developed. We postulate that this constellation of unusual features suggests a dynamic process of mechanical compromise in the posterior fossa, perhaps initiated by a rapidly growing cerebellum, which leads to congestion of the venous drainage with subsequently compromised cerebrospinal fluid reabsorption, all of which increases the posterior fossa pressure and leads to acquired tonsillar herniation. We make a distinction between congenital Chiari I malformation and acquired cerebellar tonsil herniation in this syndrome. We also observed numerous examples of abnormal cortical morphogenesis, including focal cortical dysplasia, polymicrogyria which primarily involved the perisylvian and insular regions, and cerebral and/or cerebellar asymmetric overgrowth. Other findings included a high frequency of cavum septum pellucidum or vergae, thickened corpus callosum, prominent optic nerve sheaths and a single case of venous sinus thrombosis. One patient was found to have a frontal perifalcine mass resembling a meningioma at age 5 years. This is the second apparent occurrence of this specific tumor in M–CM.

Bortezomib in the rapid reduction of high sustained antibody titers in disorders treated with therapeutic protein: lessons learned from Pompe disease

Purpose:High sustained antibody titers complicate many disorders treated with a therapeutic protein, including those treated with enzyme replacement therapy, such as Pompe disease. Although enzyme replacement therapy with alglucosidase alfa (Myozyme) in Pompe disease has improved the prognosis of this otherwise lethal disorder, patients who develop high sustained antibody titers to alglucosidase alfa enter a prolonged phase of clinical decline resulting in death despite continued enzyme replacement therapy. Clinically effective immune-tolerance induction strategies have yet to be described in the setting of an entrenched immune response characterized by high sustained antibody titers, wherein antibody-producing plasma cells play an especially prominent role.Methods:We treated three patients with infantile Pompe disease experiencing marked clinical decline due to high sustained antibody titers. To target the plasma cell source of high sustained antibody titers, a regimen based on bortezomib (Velcade) was used in combination with rituximab, methotrexate, and intravenous immunoglobulin.Results:The treatment regimen was well tolerated, with no obvious side effects. Patient 1 had a 2,048-fold, and patients 2 and 3 each had a 64-fold, reduction in anti-alglucosidase alfa antibody titer, with concomitant sustained clinical improvement.Conclusion:The addition of bortezomib to immunomodulatory regimens is an effective and safe treatment strategy in infantile Pompe disease, with potentially broader clinical implications.Genet Med 2013:15(2):123–131

PIK3CA-associated developmental disorders exhibit distinct classes of mutations with variable expression and tissue distribution

Mosaicism is increasingly recognized as a cause of developmental disorders with the advent of next-generation sequencing (NGS). Mosaic mutations of PIK3CA have been associated with the widest spectrum of phenotypes associated with overgrowth and vascular malformations. We performed targeted NGS using 2 independent deep-coverage methods that utilize molecular inversion probes and amplicon sequencing in a cohort of 241 samples from 181 individuals with brain and/or body overgrowth. We identified PIK3CA mutations in 60 individuals. Several other individuals (n = 12) were identified separately to have mutations in PIK3CA by clinical targeted-panel testing (n = 6), whole-exome sequencing (n = 5), or Sanger sequencing (n = 1). Based on the clinical and molecular features, this cohort segregated into three distinct groups: (a) severe focal overgrowth due to low-level but highly activating (hotspot) mutations, (b) predominantly brain overgrowth and less severe somatic overgrowth due to less-activating mutations, and (c) intermediate phenotypes (capillary malformations with overgrowth) with intermediately activating mutations. Sixteen of 29 PIK3CA mutations were novel. We also identified constitutional PIK3CA mutations in 10 patients. Our molecular data, combined with review of the literature, show that PIK3CA-related overgrowth disorders comprise a discontinuous spectrum of disorders that correlate with the severity and distribution of mutations.

Improved diagnostics lead to identification of three new patients with congenital disorder of glycosylation-Ip†

Congenital disorders of glycosylation (CDG) comprise a clinically and biochemically heterogeneous group of monogenetic‐inherited, multisystemic diseases that affect the biosynthesis of N‐ and/or O‐glycans linked to glycoconjugates. Recently, we identified the first patient with a defect in the cytosolic‐orientated GDP‐mannose:Man3‐4GlcNAc2‐PP‐dolichol alpha‐1,2‐mannosyltransferase (ALG11), who presented an accumulation of shortened dolichol‐linked oligosaccharides leading to CDG‐Ip (ALG11‐CDG). Here we describe an improved metabolic labeling method that allowed the identification of three new CDG‐Ip cases that were missed so far in routine diagnostics. Although all CDG‐Ip patients carry different mutations in the ALG11 gene, they share a variety of clinical syndromes like an unremarkable prenatal period followed by developmental delay, psychomotor, and mental retardation, strabismus convergens and seizures occurring in the first year of life. Hum Mutat 33:485–487, 2012.

A patient with TCIRG1-related infantile osteopetrosis presenting with congenital anomalies: Chance association or a case for pleiotropy?†

Autosomal recessive infantile osteopetrosis (ARIO) is the most clinically severe of the sclerotic bone disorders to present in the live born. This is a rare condition, estimated to affect about 1/300,000 live births. Currently, three genes are known to cause this disorder: TCIRG1 ( 50–60% of cases) encodes the a3 subunit of the osteoclast vacuolar ATPase proton pump, CLCN7 ( 15% of cases) encodes a protein of unclear function though it is thought to be an osteoclast chloride channel at the ruffled border of bone resorption, andOSTM1 (<5%of cases) is a gene whose function is poorly understood [Ramirez et al., 2004; Tolar et al., 2004]. In ARIO, the defective gene products disrupt osteoclast function by altering the normal acidification within the lacuna at the osteoclast-bone surface, thereby inhibiting normal bone resorption. Affected individuals usually present within the first year of life with failure to thrive, bone marrow failure and secondary hepatosplenomegaly with resultant anemia or thrombocytopenia, cranial nerve impingement due to stenosis of the neural foramina, hydrocephalus, hypocalcemia, and fractures. Radiographs are usually diagnostic and specific, showing diffuse osteosclerosis, poor corticomedullary differentiation and periosteal reaction. The course is aggressive and may rapidly progress, with approximately 70% of patients succumbing to complications within the first 6 years of life [Gerritsen et al., 1994]. Except for the secondary sequelae already mentioned, extra-osseous abnormalities are not normally part of the ARIO phenotype caused by mutations in either TCIRG1 or CLCN7. In contrast, patients with ARIO due to mutations in OSTM1 often suffer from a severe neurologic phenotype with abnormal muscle tone and structural brain anomalies [Pangrazio et al., 2006]. We report on a patient with ARIO caused by a previously described, homozygous mutation in the TCIRG1 gene who was initially referred for evaluation of congenital unilateral limb and kidney malformations. This male patient was the first child born to a nonconsanguineous couple of mixed Northern and Western European background (father 40 years, mother 27 years of age). The family history was unremarkable with no history of birth defects. His mother had two healthy children from a previous partner. The pregnancy history was significant for maternal minocycline exposure (for acne) during the first 2 months of gestation. Prenatal ultrasound and glucose tolerance tests were normal. Birth weight was 3,585 g ( 50%), and birth length was 21 inches (75–90%). The delivery was without complications. At birth, left limb anomalies were noted, including a fixed, pronated position at the elbow and complete cutaneous syndactyly of the 4th and 5th fingers (Fig. 1). The right arm appeared normal with full range of motion. There was also a subtle left anterior rib defect with decreased musculature of the left shoulder region. This prompted an assessment for other anomalies. Echocardiogram was normal for age (showing patent foramen ovale). Initial renal ultrasound in the newborn period identified a

132 THE FEMALE PHENOTYPE OF OTO-PALATO-DIGITAL SYNDROME TYPE 2— A REVIEW AND REPORT OF POSSIBLE NEW FEATURES

Oto-Palato-Digital (OPD) syndrome has recently been determined to be caused by mutations in Filamin A, encoded by the FLNA gene on Xq28. Filamin A is a structural protein which is expressed in diverse tissues and functions in cytoskeleton organization. Mutations in different functional domains of the protein result in similar but distinct disorders, including OPD types 1 and 2, Melnick-Needles syndrome, and frontometaphyseal dysplasia. OPD-1 was first characterized by Taybi in 1962 and over 30 cases have since been described in the literature. Principal features include broad forehead, hypertelorism, cleft palate, short stature, conductive hearing loss, and various anomalies of the hands and feet including broad (spatulate) toes, clinodactyly, syndactyly, and mild to moderate mental retardation. Female heterozygotes typically exhibit a milder phenotype with regard to all salient features, with normal intelligence, subtle facial dysmorphism, and mild involvement of the fingers or toes. OPD-2, first delineated by Fitch in 1976, presents a dramatically more severe phenotype in all respects, with frequent early lethality in affected males and a phenotype in female carriers that closely approximates males with OPD-1. Patients with OPD have been described whose phenotypes fall between the two disorders, suggesting a spectrum of severity rather than distinct clinical entities. We report a 22 year old female with dysmorphic features resembling OPD-2, including short stature, wide forehead, hypertelorism, antimongoloid slant to palpebral fissures, low-set, posteriorly rotated ears, small mandible, submucosal cleft palate with bifid uvula, clinodactyly and camptodactyly, broad distal digits, and flexion contractures. Radiographs reveal Klippel-Feil anomaly and spondylolysis of L5-S1, uncommon but reported findings in this condition. This patient also exhibits marked keloid formation at surgical scars, focal myocardial hypertrophy which necessitated surgical resection, and a bicornuate uterus. Neither the tissue overgrowth, nor the uterine malformation has been described previously in reported cases of females with OPD. A review of the literature will be presented with comparison of the findings in this patient to those in other reported females. Data on mutational analysis in females with this disorder is limited to date. Molecular analysis is planned and will be reported if available.