Fragile X syndrome (FXS) is caused by a full mutation expansion (>200 CGG repeats) in the FMR1 gene that results in a deficiency of the fragile X mental retardation protein. Although most individuals with the premutation (55-200 CGG repeats) are considered unaffected by FXS, recent case studies have documented children with the premutation who have cognitive deficits, behavioral problems, and/or autism spectrum disorders. The objective of this study was to compare the prevalence of autism spectrum disorders (ASD) and attention-deficit hyperactivity disorder (ADHD) symptoms in boys with the premutation who presented as probands, in brothers with the premutation who did not present as probands, and in normal brothers of premutation and/or full mutation carriers. Participants included 43 male children: 14 probands who presented to clinic, 13 nonprobands who were identified through cascade testing (routine genetic testing of family members after identification of a proband) and confirmed to have the premutation, and a control group of 16 male siblings of individuals with the fragile X premutation or full mutation who were negative for the FMR1 mutation. Participants came from 1 of 2 collaborative sites: University of California, Davis and La Trobe University in Australia. Parents completed the Conners' Global Index-Parent Version for assessing symptoms of ADHD and the Social Communication Questionnaire (SCQ) for identifying symptoms of ASD. Children who were in the ASD range on the SCQ (n = 13) underwent further evaluation with either the Autism Diagnostic Observation Schedule-Generic (n = 10) or the Autism Diagnostic Interview-Revised (n = 3). A final diagnosis of ASD included clinical assessment utilizing DSM-IV-TR criteria in addition to the standardized assessments. There was a higher rate of ASD in boys with the premutation presenting as probands (p < 0.001) or nonprobands (p < .04) compared with sibling controls without the premutation. In addition, probands had a significant increase in ADHD symptoms compared with controls (p < .0001). Of the probands, 93% had symptoms of ADHD and 79% had ASD. In the nonproband premutation group, 38% had symptoms of ADHD and 8% had ASD. Thirteen percent of sibling controls had symptoms of ADHD and none had ASD. IQ scores were similar in all 3 groups (p = .13), but the use of psychotropic medications was significantly higher in probands with the premutation compared with that in controls (p < .0001). Developmental problems have been observed in premutation carriers, particularly those who present clinically with behavioral difficulties. Although this study is based on a small sample size, it suggests that premutation carriers, even those who do not present clinically, may be at increased risk for an ASD and/or symptoms of ADHD. If the premutation is identified through cascade testing, then further assessment should be carried out for symptoms of ADHD, social deficits, or learning disabilities.

SATB2-associated syndrome (SAS) is an autosomal dominant disorder characterized by significant neurodevelopmental disabilities with limited to absent speech, behavioral issues, and craniofacial anomalies. Previous studies have largely been restricted to case reports and small series without in-depth phenotypic characterization or genotype-phenotype correlations. Seventy two study participants were identified as part of the SAS clinical registry. Individuals with a molecularly confirmed diagnosis of SAS were referred after clinical diagnostic testing. In this series we present the most comprehensive phenotypic and genotypic characterization of SAS to date, including prevalence of each clinical feature, neurodevelopmental milestones, and when available, patient management. We confirm that the most distinctive features are neurodevelopmental delay with invariably severely limited speech, abnormalities of the palate (cleft or high-arched), dental anomalies (crowding, macrodontia, abnormal shape), and behavioral issues with or without bone or brain anomalies. This comprehensive clinical characterization will help clinicians with the diagnosis, counseling and management of SAS and help provide families with anticipatory guidance.

The SATB2-associated syndrome (SAS) was recently proposed as a clinically recognizable syndrome that results from deleterious alterations of the SATB2 gene in humans. Although interstitial deletions at 2q33 encompassing SATB2, either alone or contiguously with other genes, have been reported before, there is limited literature regarding intragenic mutations of this gene and the resulting phenotype. We describe five patients in whom whole exome sequencing identified five unique de novo mutations in the SATB2 gene (one splice site, one frameshift, and three nonsense mutations). The five patients had overlapping features that support the characteristic features of the SAS: intellectual disability with limited speech development and craniofacial abnormalities including cleft palate, dysmorphic features, and dental abnormalities. Furthermore, Patient 1 also had features not previously described that represent an expansion of the phenotype. Osteopenia was seen in two of the patients, suggesting that this finding could be added to the list of distinctive findings. We provide supporting evidence that analysis for deletions or point mutations in SATB2 should be considered in children with intellectual disability and severely impaired speech, cleft or high palate, teeth abnormalities, and osteopenia.

Cleft lip and/or palate (CL/P) are common structural birth defects in humans. We used exome sequencing to study a patient with bilateral CL/P and identified a single nucleotide deletion in the patient and her similarly affected son—c.546_546delG, predicting p.Gln183Argfs*57 in the Distal-less 4 (DLX4) gene. The sequence variant was absent from databases, predicted to be deleterious and was verified by Sanger sequencing. In mammals, there are three Dlx homeobox clusters with closely located gene pairs (Dlx1/Dlx2, Dlx3/Dlx4, Dlx5/Dlx6). In situ hybridization showed that Dlx4 was expressed in the mesenchyme of the murine palatal shelves at E12.5, prior to palate closure. Wild-type human DLX4, but not mutant DLX4_c.546delG, could activate two murine Dlx conserved regulatory elements, implying that the mutation caused haploinsufficiency. We showed that reduced DLX4 expression after short interfering RNA treatment in a human cell line resulted in significant up-regulation of DLX3, DLX5 and DLX6, with reduced expression of DLX2 and significant up-regulation of BMP4, although the increased BMP4 expression was demonstrated only in HeLa cells. We used antisense morpholino oligonucleotides to target the orthologous Danio rerio gene, dlx4b, and found reduced cranial size and abnormal cartilaginous elements. We sequenced DLX4 in 155 patients with non-syndromic CL/P and CP, but observed no sequence variants. From the published literature, Dlx1/Dlx2 double homozygous null mice and Dlx5 homozygous null mice both have clefts of the secondary palate. This first finding of a DLX4 mutation in a family with CL/P establishes DLX4 as a potential cause of human clefts.

We report on the occurrence of congenital diaphragmatic hernia in a family with craniofrontonasal syndrome found to have a previously unreported mutation in EFNB1. The female proband presented with hypertelorism, telecanthus, bifid nasal tip, widow's peak, frontal bossing, and a widened metopic suture. Her father was noted to have hypertelorism, telecanthus, widow's peak, and a history of pectus carinatum. He was found to have a previously unreported mutation in exon 5 of EFNB1 predicted to cause premature protein truncation. The parents of the proband previously had a female fetus with congenital diaphragmatic hernia. The occurrence of congenital diaphragmatic hernia, phenotypic differences between males and females, and utility of molecular testing in craniofrontonasal syndrome are demonstrated.

Despite major progress in defining the genetic basis of Mendelian disorders, the molecular etiology of many cases remains unknown. Patients with these undiagnosed disorders often have complex presentations and require treatment by multiple health care specialists. Here, we describe an integrated clinical diagnostic and research program using whole-exome and whole-genome sequencing (WES/WGS) for Mendelian disease gene discovery. This program employs specific case ascertainment parameters, a WES/WGS computational analysis pipeline that is optimized for Mendelian disease gene discovery with variant callers tuned to specific inheritance modes, an interdisciplinary crowdsourcing strategy for genomic sequence analysis, matchmaking for additional cases, and integration of the findings regarding gene causality with the clinical management plan. The interdisciplinary gene discovery team includes clinical, computational, and experimental biomedical specialists who interact to identify the genetic etiology of the disease, and when so warranted, to devise improved or novel treatments for affected patients. This program effectively integrates the clinical and research missions of an academic medical center and affords both diagnostic and therapeutic options for patients suffering from genetic disease. It may therefore be germane to other academic medical institutions engaged in implementing genomic medicine programs.

Current genetic tests for rare diseases provide a diagnosis in only a modest proportion of cases. The Full Genome Analysis method, FGA, combines long-range assembly and whole-genome sequencing to detect small variants, structural variants with breakpoint resolution, and phasing. We built a variant prioritization pipeline and tested FGAs utility for diagnosis of rare diseases in a clinical setting. FGA identified structural variants and small variants with an overall diagnostic yield of 40% (20 of 50 cases) and 35% in exome-negative cases (8 of 23 cases), 4 of these were structural variants. FGA detected and mapped structural variants that are missed by short reads, including non-coding duplication, and phased variants across long distances of more than 180kb. With the prioritization algorithm, longer DNA technologies could replace multiple tests for monogenic disorders and expand the range of variants detected. Our study suggests that genomes produced from technologies like FGA can improve variant detection and provide higher resolution genome maps for future application.

Beare-Stevenson syndrome (BSS; OMIM 123790) is a rare craniosynostosis syndrome characterized by cutis gyrata, acanthosis nigricans, skin tags, anogenital anomalies and a prominent umbilicus. Two activating point mutations in FGFR2, p.Y375C and p.S372C, account for 50-60% of patients with BSS, and locus and/or allelic heterogeneity have been proposed. We present a novel deletion of 21 amino acids in the FGFR2 gene, c.1506del63, in a patient with BSS. BSS results from a gain of FGFR2 function, and this deletion may act by altering the splicing of isoform IIIc, resulting in illegitimate expression and thus a gain of function of FGFR2b.The propositus (Fig. 1) was delivered by cesarean for a dizygotic twin pregnancy at term to a 40-year-old, G3P2-4 mother and a 48-year-old father. Birth parameters were weight 3,860 g (75-90th centile for a singleton male), length 51 cm (50-75th centile for a singleton male) and head circumference 36.5 cm (75-90th centile for a singleton male). Striking craniofacial features were present, including ocular proptosis with hypoplasia of the supraorbital ridges, hypertelorism with a divergent strabismus, deep creases below his eyes, a high nasal bridge, midface hypoplasia and complete atresia of the external ear canals with a left preauricular pit and bilateral ear creases. There were multiple neonatal teeth, gingival hyperplasia and a high-arched, narrow palate with a bifid uvula. Cutaneous features included cutis gyrata and acanthosis nigricans of the posterior scalp, small skin tags at the corners of the mouth, excess neck skin and a prominent umbilicus with redundant skin. A sacral tag, hypospadias and a prominent scrotal raph e were present. Examination of the hands and feet showed redundant palmar and plantar skin, slightly broad thumbs and halluces and mild skin syndactyly of the second and third toes with overlapping fourth toes. Investigations for a flat occiput and a prominent forehead at 4 months of age showed fusion of the posterior aspect of the sagittal and the proximal lambdoid sutures and cranial vault remodeling was performed at 7 months. Bilateral choanal stenosis and a mild to moderate mixed hearing loss requiring hearing aids were present. Ophthalmological examination detected mild optic nerve hypoplasia and high myopia with astigmatism requiring corrective lenses. Obstructive sleep apnea was treated with a tracheostomy and a ventriculoperitoneal shunt was placed because of an ArnoldChiari malformation with hydrocephalus. His length was 107 cm at age 6 years (3rd centile), but his other growth parameters were normal. He has had initial mild motor delays, but the propositus' neurodevelopmental progress was normal.A clinical diagnosis of BSS was made shortly after birth. At that time, targeted re-sequencing for the two previously known FGFR2 mutations, p.Y375 C and p.S372 C , was performed on DNA extracted from peripheral blood lymphocytes from the propositus, and was negative.The diagnosis of a rhabdomyosarcoma in his twin when both were 5 years of age prompted further ...