The genomic landscape of balanced cytogenetic abnormalities associated with human congenital anomalies.

Despite the clinical significance of balanced chromosomal abnormalities (BCAs), their characterization has largely been restricted to cytogenetic resolution. We explored the landscape of BCAs at nucleotide resolution in 273 subjects with a spectrum of congenital anomalies. Whole-genome sequencing revised 93% of karyotypes and demonstrated complexity that was cryptic to karyotyping in 21% of BCAs, highlighting the limitations of conventional cytogenetic approaches. At least 33.9% of BCAs resulted in gene disruption that likely contributed to the developmental phenotype, 5.2% were associated with pathogenic genomic imbalances, and 7.3% disrupted topologically associated domains (TADs) encompassing known syndromic loci. Remarkably, BCA breakpoints in eight subjects altered a single TAD encompassing MEF2C, a known driver of 5q14.3 microdeletion syndrome, resulting in decreased MEF2C expression. We propose that sequence-level resolution dramatically improves prediction of clinical outcomes for balanced rearrangements and provides insight into new pathogenic mechanisms, such as altered regulation due to changes in chromosome topology.

Actin capping protein CAPZB regulates cell morphology, differentiation, and neural crest migration in craniofacial morphogenesis†.

CAPZB is an actin-capping protein that caps the growing end of F-actin and modulates the cytoskeleton and tethers actin filaments to the Z-line of the sarcomere in muscles. Whole-genome sequencing was performed on a subject with micrognathia, cleft palate and hypotonia that harbored a de novo, balanced chromosomal translocation that disrupts the CAPZB gene. The function of capzb was analyzed in the zebrafish model. capzb(-/-) mutants exhibit both craniofacial and muscle defects that recapitulate the phenotypes observed in the human subject. Loss of capzb affects cell morphology, differentiation and neural crest migration. Differentiation of both myogenic stem cells and neural crest cells requires capzb. During palate morphogenesis, defective cranial neural crest cell migration in capzb(-/-) mutants results in loss of the median cell population, creating a cleft phenotype. capzb is also required for trunk neural crest migration, as evident from melanophores disorganization in capzb(-/-) mutants. In addition, capzb over-expression results in embryonic lethality. Therefore, proper capzb dosage is important during embryogenesis, and regulates both cell behavior and tissue morphogenesis.

High Incidence of Noonan Syndrome Features Including Short Stature and Pulmonic Stenosis in Patients carrying NF1 Missense Mutations Affecting p.Arg1809: Genotype-Phenotype Correlation.

Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype-phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple café-au-lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan-like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P < 0.0001). Developmental delays and/or learning disabilities were reported in over 50% of patients. Melanocytes cultured from a CALM in a segmental NF1-patient showed two different somatic NF1 mutations, p.Arg1809Cys and a multi-exon deletion, providing genetic evidence that p.Arg1809Cys is a loss-of-function mutation in the melanocytes and causes a pigmentary phenotype. Constitutional missense mutations at p.Arg1809 affect 1.23% of unrelated NF1 probands in the UAB cohort, therefore this specific NF1 genotype-phenotype correlation will affect counseling and management of a significant number of patients.

A case of an atypically large proximal 15q deletion as cause for Prader-Willi syndrome arising from a de novo unbalanced translocation.

We describe an 11 month old female with Prader-Willi syndrome (PWS) resulting from an atypically large deletion of proximal 15q due to a de novo 3;15 unbalanced translocation. The 10.6 Mb deletion extends from the chromosome 15 short arm and is not situated in a region previously reported as a common distal breakpoint for unbalanced translocations. There was no deletion of the reciprocal chromosome 3q subtelomeric region detected by either chromosomal microarray or FISH. The patient has hypotonia, failure to thrive, and typical dysmorphic facial features for PWS. The patient also has profound global developmental delay consistent with an expanded, more severe, phenotype.

Multigeneration family with short stature, developmental delay, and dysmorphic features due to 4q27-q28.1 microdeletion.

Deletions of the long arm of chromosome 4 are rare but have been previously reported to be associated with craniofacial anomalies, digital anomalies, developmental delay, growth failure, and cardiovascular anomalies. Strehle et al. previously presented 20 patients with 4q deletions and began to construct a phenotype-genotype map for chromosome 4q. This report follows up on that work by providing clinical and molecular cytogenetic data on a three generation pedigree including seven patients with short stature, dysmorphic features, and developmental delay identified to have a 4q27-q28.1 microdeletion of approximately 5.68 Mb by oligonucleotide chromosomal microarray. This family represents a rare report of an inherited interstitial deletion of the long arm of chromosome 4. To our knowledge, only two cases have been previously reported. The contribution of candidate genes in the region is discussed.

Microarray comparative genomic hybridization and cytogenetic characterization of tissue-specific mosaicism in three patients.

The presence of more than one cell line in an individual may often be missed by classical cytogenetic analysis due to a low percentage of affected cells or analysis of cells from an unaffected or less affected germ layer. Array comparative genomic hybridization (aCGH) from whole blood or tissue is an important adjunct to standard karyotyping due to its ability to detect genomic imbalances that are below the resolution of karyotype analysis. We report results from three unrelated patients in whom aCGH revealed mosaicism not identified by peripheral blood chromosome analysis. This study further illustrates the important application of aCGH in detecting tissue-specific mosaicism, thereby leading to an improvement in the ability to provide a diagnosis for patients with normal chromosome analysis and dysmorphic features, congenital anomalies, and/or developmental delay.

De novo mutations in the actin genes ACTB and ACTG1 cause Baraitser-Winter syndrome.

Brain malformations are individually rare but collectively common causes of developmental disabilities. Many forms of malformation occur sporadically and are associated with reduced reproductive fitness, pointing to a causative role for de novo mutations. Here, we report a study of Baraitser-Winter syndrome, a well-defined disorder characterized by distinct craniofacial features, ocular colobomata and neuronal migration defect. Using whole-exome sequencing of three proband-parent trios, we identified de novo missense changes in the cytoplasmic actin-encoding genes ACTB and ACTG1 in one and two probands, respectively. Sequencing of both genes in 15 additional affected individuals identified disease-causing mutations in all probands, including two recurrent de novo alterations (ACTB, encoding p.Arg196His, and ACTG1, encoding p.Ser155Phe). Our results confirm that trio-based exome sequencing is a powerful approach to discover genes causing sporadic developmental disorders, emphasize the overlapping roles of cytoplasmic actin proteins in development and suggest that Baraitser-Winter syndrome is the predominant phenotype associated with mutation of these two genes.

Microdeletion 9q22.3 syndrome includes metopic craniosynostosis, hydrocephalus, macrosomia, and developmental delay.

Basal cell nevus syndrome (BCNS), also known as Gorlin syndrome (OMIM #109400) is a well-described rare autosomal dominant condition due to haploinsufficiency of PTCH1. With the availability of comparative genomic hybridization arrays, increasing numbers of individuals with microdeletions involving this locus are being identified. We present 10 previously unreported individuals with 9q22.3 deletions that include PTCH1. While 7 of the 10 patients (7 females, 3 males) did not meet strict clinical criteria for BCNS at the time of molecular diagnosis, almost all of the patients were too young to exhibit many of the diagnostic features. A number of the patients exhibited metopic craniosynostosis, severe obstructive hydrocephalus, and macrosomia, which are not typically observed in BCNS. All individuals older than a few months of age also had developmental delays and/or intellectual disability. Only facial features typical of BCNS, except in those with prominent midforeheads secondary to metopic craniosynostosis, were shared among the 10 patients. The deletions in these individuals ranged from 352 kb to 20.5 Mb in size, the largest spanning 9q21.33 through 9q31.2. There was significant overlap of the deleted segments among most of the patients. The smallest common regions shared among the deletions were identified in order to localize putative candidate genes that are potentially responsible for each of the non-BCNS features. These were a 929 kb region for metopic craniosynostosis, a 1.08 Mb region for obstructive hydrocephalus, and a 1.84 Mb region for macrosomia. Additional studies are needed to further characterize the candidate genes within these regions.

Unexpected detection of dystrophin gene deletions by array comparative genomic hybridization.

Array comparative genomic hybridization has increasingly become the standard of care to evaluate patients for genomic imbalance. As the patient population evaluated by microarray expands, there is certain to be an increase in the detection of unexpected, yet common diseases. When array results predict a late-onset disorder or cancer predisposition, it becomes a challenge for physicians and counselors to adequately address with patients. Included in this study were three patients described with nonspecific phenotypic findings who underwent microarray testing to better define their disease etiology. An unexpected deletion within the dystrophin gene was observed in each case, despite that no patient was suspected of a dystrophinopathy at the time of testing. The patients included an 8-day-old male with a dystrophin deletion predictive of Becker muscular dystrophy, an 18-month old female found to be the carrier of deletion, and a 4-year-8-month-old male with a deletion predictive of Duchenne muscular dystrophy. In this circumstance it becomes difficult to counsel the family, as well as to predict disease course when underlying medical conditions may exist. However, early detection may enable the patient to receive proactive treatment, and allows for screening of at-risk family members. Ultimately, it is up to the clinician to promote informed decision-making within the family prior to testing, and ensure that adequate counseling is provided during follow-up.

Confirmation study of PTEN mutations among individuals with autism or developmental delays/mental retardation and macrocephaly.

There is a strong genetic component to autism spectrum disorders (ASD), but due to significant genetic heterogeneity, individual genetic abnormalities contribute a small percentage to the overall total. Previous studies have demonstrated PTEN mutations in a sizable proportion of individuals with ASD or mental retardation/developmental delays (MR/DD) and macrocephaly that do not have features of Cowden or Bannayan-Riley-Ruvalcaba syndrome. This study was performed to confirm our previous results. We reviewed the charts of individuals who had PTEN clinical sequencing performed at our institution from January 2008 to July 2009. There were 93 subjects tested from our institution during that period. PTEN mutations were found in 2/39 (5.1%) ASD patients and 2/51 (3.9%) MR/DD patients. Three additional patients without mutations had no diagnostic information. Multiple relatives of individuals with a PTEN mutation had macrocephaly, MR, or early onset cancer (breast, renal, and prostate). Of those relatives tested, all had the familial PTEN mutation. None of the affected relatives had previously been diagnosed with Cowden or Bannayan-Riley-Ruvalcaba syndrome. We noted in our previous study several adult relatives without any findings who carried a mutation. Combined with data from our previous cohort, we have found PTEN mutations in 7/99 (7.1%) of individuals with ASD and 8/100 (8.0%) of individuals with MR/DD, all of whom had macrocephaly. We recommend testing for mutations in PTEN for individuals with ASD or MR/DD and macrocephaly. If mutations are found, other family members should be offered testing and the adults offered cancer screening if they have a PTEN mutation.

Identification of familial and de novo microduplications of 22q11.21-q11.23 distal to the 22q11.21 microdeletion syndrome region.

Deletions of the 22q11.2 region distal to the 22q11.21 microdeletion syndrome region have recently been described in individuals with mental retardation and congenital anomalies. Because these deletions are mediated by low-copy repeats (LCRs), located distal to the 22q11.21 DiGeorge/velocardiofacial microdeletion region, duplications are predicted to occur with a frequency equal to the deletion. However, few microduplications of this region have been reported. We report the identification of 18 individuals with microduplications of 22q11.21-q11.23. The duplication boundaries for all individuals are within LCRs distal to the DiGeorge/velocardiofacial microdeletion region. Clinical records for nine subjects reveal shared characteristics, but also several examples of contradicting clinical features (e.g. macrocephaly versus microcephaly and upslanting versus downslanting palpebral fissures). Of 12 cases for whom parental DNA samples were available for testing, one is de novo and 11 inherited the microduplication from a parent, three of whom reportedly have learning problems or developmental delay. The variable phenotypes and preponderance of familial cases obfuscate the clinical relevance of the molecular data and emphasize the need for careful parental assessments and clinical correlations.

Neonatal cyanosis due to a novel fetal hemoglobin: Hb F-Circleville [Ggamma63(E7)His-->Leu, CAT>CTT].

Neonatal cyanosis can result from a multitude of acquired and inherited causes. Cyanosis resulting from fetal M hemoglobin (Hb) variants is very rare. Only two (G)gamma variants causing methemoglobinemia and cyanosis in the newborn have been reported to date. Here we describe a novel fetal Hb variant, Hb F-Circleville [Ggamma63(E7)His-->Leu], associated with methemoglobinemia and cyanosis in the newborn. The patient's sister also had neonatal cyanosis at birth.

X-linked infantile periodic alternating nystagmus.

OBJECTIVE: To describe the clinical and electrophysiological characterization of four family members from three generations who have X-linked infantile periodic alternating nystagmus (XIPAN). METHODS: Complete clinical ophthalmological evaluation, pedigree analysis, electroretinograms (ERG), eye movement recordings (EMR), color vision, and fundus photography were performed on all subjects. RESULTS: Three males in two generations and one female were examined. Clinical examinations showed a jerk/pendular nystagmus with a latent component, strabismus, and a significant refractive error in the three affected males, while the female had only myopic astigmatism. ERG, color contrast, and fundus examinations were normal in all four family members. All four family members showed EMR abnormalities with infantile jerk/dual jerk and pendular nystagmus waveforms. The female had nystagmus present on EMR only and all patients showed (a)periodicity to their nystagmus. CONCLUSIONS: In this family with no other congenital visual sensory system disease, affected males had obvious periodic alternating nystagmus, strabismus, and refractive errors, while the female had clinically "silent" periodic nystagmus that is probably a marker for the carrier state.

Leigh disease with mitochondrial DNA A8344G mutation: case report and brief review.

Leigh disease, subacute necrotizing encephalomyelopathy, is a neurodegenerative disorder often seen in infancy or childhood but rarely reported in adults. Genetic heterogeneity is well recognized, and the associated etiologies include both mitochondrial and nuclear DNA defects. We describe an infant presenting with developmental delay and then progressive multisystem disorder and neuroradiologic features of Leigh disease. He and his maternal relatives all have the A8344G mitochondrial DNA mutation. However, only minor clinical features are seen in his maternal relatives, with migraine being the most common problem. Additionally the A8344G mitochondrial DNA mutation is associated with spinocerebellar degeneration, other nonspecific mitochondrial encephalomyopathies, atypical Charcot-Marie-Tooth disease, and progressive external ophthalmoplegia. The A8344G mitochondrial DNA mutation may present with Leigh disease or other different atypical clinical features without myoclonic epilepsy and ragged red fibers.