A dyadic approach to the delineation of diagnostic entities in clinical genomics.

The delineation of disease entities is complex, yet recent advances in the molecular characterization of diseases provide opportunities to designate diseases in a biologically valid manner. Here, we have formalized an approach to the delineation of Mendelian genetic disorders that encompasses two distinct but inter-related concepts: (1) the gene that is mutated and (2) the phenotypic descriptor, preferably a recognizably distinct phenotype. We assert that only by a combinatorial or dyadic approach taking both of these attributes into account can a unitary, distinct genetic disorder be designated. We propose that all Mendelian disorders should be designated as "GENE-related phenotype descriptor" (e.g., "CFTR-related cystic fibrosis"). This approach to delineating and naming disorders reconciles the complexity of gene-to-phenotype relationships in a simple and clear manner yet communicates the complexity and nuance of these relationships.

5q35 duplication presents with psychiatric and undergrowth phenotypes mediated by NSD1 overexpression and mTOR signaling downregulation.

PURPOSE: Nuclear receptor binding SET domain protein 1, NSD1, encodes a histone methyltransferase H3K36. NSD1 is responsible for the phenotype of the reciprocal 5q35.2q35.3 microdeletion-microduplication syndromes. We expand the phenotype and demonstrate the functional role of NSD1 in microduplication 5q35 syndrome. METHODS: Through an international collaboration, we report nine new patients, contributing to the emerging phenotype, highlighting psychiatric phenotypes in older affected individuals. Focusing specifically on the undergrowth phenotype, we have modeled the effects of Mes-4/NSD overexpression in Drosophila melanogaster. RESULTS: The individuals (including a family) from diverse backgrounds with duplications ranging in size from 0.6 to 4.5 Mb, have a consistent undergrowth phenotype. Mes-4 overexpression in the developing wing causes undergrowth, increased H3K36 methylation, and increased apoptosis. We demonstrate that altering the levels of insulin receptor (IR) rescues the apoptosis and the wing undergrowth phenotype, suggesting changes in mTOR pathway signaling. Leucine supplementation rescued Mes-4/NSD induced cell death, demonstrating decreased mTOR signaling caused by NSD1. CONCLUSION: Given that we show mTOR inhibition as a likely mechanism and amelioration of the phenotype by leucine supplementation in a fly model, we suggest further studies should evaluate the therapeutic potential of leucine or branched chain amino acids as an adjunct possible treatment to ameliorate human growth and psychiatric phenotypes and propose inclusion of 5q35-microduplication as part of the differential diagnosis for children and adults with delayed bone age, short stature, microcephaly, developmental delay, and psychiatric phenotypes.

Mosaic Activating Mutations in FGFR1 Cause Encephalocraniocutaneous Lipomatosis.

Encephalocraniocutaneous lipomatosis (ECCL) is a sporadic condition characterized by ocular, cutaneous, and central nervous system anomalies. Key clinical features include a well-demarcated hairless fatty nevus on the scalp, benign ocular tumors, and central nervous system lipomas. Seizures, spasticity, and intellectual disability can be present, although affected individuals without seizures and with normal intellect have also been reported. Given the patchy and asymmetric nature of the malformations, ECCL has been hypothesized to be due to a post-zygotic, mosaic mutation. Despite phenotypic overlap with several other disorders associated with mutations in the RAS-MAPK and PI3K-AKT pathways, the molecular etiology of ECCL remains unknown. Using exome sequencing of DNA from multiple affected tissues from five unrelated individuals with ECCL, we identified two mosaic mutations, c.1638C>A (p.Asn546Lys) and c.1966A>G (p.Lys656Glu) within the tyrosine kinase domain of FGFR1, in two affected individuals each. These two residues are the most commonly mutated residues in FGFR1 in human cancers and are associated primarily with CNS tumors. Targeted resequencing of FGFR1 in multiple tissues from an independent cohort of individuals with ECCL identified one additional individual with a c.1638C>A (p.Asn546Lys) mutation in FGFR1. Functional studies of ECCL fibroblast cell lines show increased levels of phosphorylated FGFRs and phosphorylated FRS2, a direct substrate of FGFR1, as well as constitutive activation of RAS-MAPK signaling. In addition to identifying the molecular etiology of ECCL, our results support the emerging overlap between mosaic developmental disorders and tumorigenesis.

Rare variants in the genetic background modulate cognitive and developmental phenotypes in individuals carrying disease-associated variants.

PURPOSE: To assess the contribution of rare variants in the genetic background toward variability of neurodevelopmental phenotypes in individuals with rare copy-number variants (CNVs) and gene-disruptive variants. METHODS: We analyzed quantitative clinical information, exome sequencing, and microarray data from 757 probands and 233 parents and siblings who carry disease-associated variants. RESULTS: The number of rare likely deleterious variants in functionally intolerant genes ("other hits") correlated with expression of neurodevelopmental phenotypes in probands with 16p12.1 deletion (n=23, p=0.004) and in autism probands carrying gene-disruptive variants (n=184, p=0.03) compared with their carrier family members. Probands with 16p12.1 deletion and a strong family history presented more severe clinical features (p=0.04) and higher burden of other hits compared with those with mild/no family history (p=0.001). The number of other hits also correlated with severity of cognitive impairment in probands carrying pathogenic CNVs (n=53) or de novo pathogenic variants in disease genes (n=290), and negatively correlated with head size among 80 probands with 16p11.2 deletion. These co-occurring hits involved known disease-associated genes such as SETD5, AUTS2, and NRXN1, and were enriched for cellular and developmental processes. CONCLUSION: Accurate genetic diagnosis of complex disorders will require complete evaluation of the genetic background even after a candidate disease-associated variant is identified.

Chitayat-Hall and Schaaf-Yang syndromes:a common aetiology: expanding the phenotype of MAGEL2-related disorders.

BACKGROUND: Chitayat-Hall syndrome, initially described in 1990, is a rare condition characterised by distal arthrogryposis, intellectual disability, dysmorphic features and hypopituitarism, in particular growth hormone deficiency. The genetic aetiology has not been identified. METHODS AND RESULTS: We identified three unrelated families with a total of six affected patients with the clinical manifestations of Chitayat-Hall syndrome. Through whole exome or whole genome sequencing, pathogenic variants in the MAGEL2 gene were identified in all affected patients. All disease-causing sequence variants detected are predicted to result in a truncated protein, including one complex variant that comprised a deletion and inversion. CONCLUSIONS: Chitayat-Hall syndrome is caused by pathogenic variants in MAGEL2 and shares a common aetiology with the recently described Schaaf-Yang syndrome. The phenotype of MAGEL2-related disorders is expanded to include growth hormone deficiency as an important and treatable complication.

De novo mutations in NALCN cause a syndrome characterized by congenital contractures of the limbs and face, hypotonia, and developmental delay.

Freeman-Sheldon syndrome, or distal arthrogryposis type 2A (DA2A), is an autosomal-dominant condition caused by mutations in MYH3 and characterized by multiple congenital contractures of the face and limbs and normal cognitive development. We identified a subset of five individuals who had been putatively diagnosed with "DA2A with severe neurological abnormalities" and for whom congenital contractures of the limbs and face, hypotonia, and global developmental delay had resulted in early death in three cases; this is a unique condition that we now refer to as CLIFAHDD syndrome. Exome sequencing identified missense mutations in the sodium leak channel, non-selective (NALCN) in four families affected by CLIFAHDD syndrome. We used molecular-inversion probes to screen for NALCN in a cohort of 202 distal arthrogryposis (DA)-affected individuals as well as concurrent exome sequencing of six other DA-affected individuals, thus revealing NALCN mutations in ten additional families with "atypical" forms of DA. All 14 mutations were missense variants predicted to alter amino acid residues in or near the S5 and S6 pore-forming segments of NALCN, highlighting the functional importance of these segments. In vitro functional studies demonstrated that NALCN alterations nearly abolished the expression of wild-type NALCN, suggesting that alterations that cause CLIFAHDD syndrome have a dominant-negative effect. In contrast, homozygosity for mutations in other regions of NALCN has been reported in three families affected by an autosomal-recessive condition characterized mainly by hypotonia and severe intellectual disability. Accordingly, mutations in NALCN can cause either a recessive or dominant condition characterized by varied though overlapping phenotypic features, perhaps based on the type of mutation and affected protein domain(s).

Molecular characterization of NRXN1 deletions from 19,263 clinical microarray cases identifies exons important for neurodevelopmental disease expression.

PURPOSE: The purpose of the current study was to assess the penetrance of NRXN1 deletions. METHODS: We compared the prevalence and genomic extent of NRXN1 deletions identified among 19,263 clinically referred cases to that of 15,264 controls. The burden of additional clinically relevant copy-number variations (CNVs) was used as a proxy to estimate the relative penetrance of NRXN1 deletions. RESULTS: We identified 41 (0.21%) previously unreported exonic NRXN1 deletions ascertained for developmental delay/intellectual disability that were significantly greater than in controls (odds ratio (OR) = 8.14; 95% confidence interval (CI): 2.91-22.72; P < 0.0001). Ten (22.7%) of these had a second clinically relevant CNV. Subjects with a deletion near the 3' end of NRXN1 were significantly more likely to have a second rare CNV than subjects with a 5' NRXN1 deletion (OR = 7.47; 95% CI: 2.36-23.61; P = 0.0006). The prevalence of intronic NRXN1 deletions was not statistically different between cases and controls (P = 0.618). The majority (63.2%) of intronic NRXN1 deletion cases had a second rare CNV at a prevalence twice as high as that for exonic NRXN1 deletion cases (P = 0.0035). CONCLUSIONS: The results support the importance of exons near the 5' end of NRXN1 in the expression of neurodevelopmental disorders. Intronic NRXN1 deletions do not appear to substantially increase the risk for clinical phenotypes.Genet Med 19 1, 53-61.

A novel multisystem disease associated with recessive mutations in the tyrosyl-tRNA synthetase (YARS) gene.

Aminoacyl-tRNA synthetases (ARSs) are a group of ubiquitously expressed enzymes that are best known for their function in the first step of protein translation but have been increasingly associated with secondary functions including transcription and translation control and extracellular signaling. Mutations in numerous ARSs have been linked to a growing number of both autosomal dominant and autosomal recessive human diseases. The tyrosyl-tRNA synthetase (YARS) links the amino acid tyrosine to its cognate tRNA. We report two siblings who presented with failure to thrive (FTT), hypertriglyceridemia, developmental delay, liver dysfunction, lung cysts, and abnormal subcortical white matter. Using exome sequencing the siblings were found to harbor bi-allelic pathogenic-appearing variants within the YARS gene (NM_003680.3):c.638C>T p.(Pro213Leu) and c.1573G>A p.(Gly525Arg). These YARS variants occur in the catalytic domain and the C-terminal domain, respectively. Mutations in YARS have been previously associated with an autosomal dominant form of Charcot-Marie-Tooth (CMT); our findings suggest the disease spectrum associated with YARS dysregulation is broader than peripheral neuropathy. © 2016 Wiley Periodicals, Inc.

SCRIB and PUF60 are primary drivers of the multisystemic phenotypes of the 8q24.3 copy-number variant.

Copy-number variants (CNVs) represent a significant interpretative challenge, given that each CNV typically affects the dosage of multiple genes. Here we report on five individuals with coloboma, microcephaly, developmental delay, short stature, and craniofacial, cardiac, and renal defects who harbor overlapping microdeletions on 8q24.3. Fine mapping localized a commonly deleted 78 kb region that contains three genes: SCRIB, NRBP2, and PUF60. In vivo dissection of the CNV showed discrete contributions of the planar cell polarity effector SCRIB and the splicing factor PUF60 to the syndromic phenotype, and the combinatorial suppression of both genes exacerbated some, but not all, phenotypic components. Consistent with these findings, we identified an individual with microcephaly, short stature, intellectual disability, and heart defects with a de novo c.505C>T variant leading to a p.His169Tyr change in PUF60. Functional testing of this allele in vivo and in vitro showed that the mutation perturbs the relative dosage of two PUF60 isoforms and, subsequently, the splicing efficiency of downstream PUF60 targets. These data inform the functions of two genes not associated previously with human genetic disease and demonstrate how CNVs can exhibit complex genetic architecture, with the phenotype being the amalgam of both discrete dosage dysfunction of single transcripts and also of binary genetic interactions.

Mutations in SRCAP, encoding SNF2-related CREBBP activator protein, cause Floating-Harbor syndrome.

Floating-Harbor syndrome (FHS) is a rare condition characterized by short stature, delayed osseous maturation, expressive-language deficits, and a distinctive facial appearance. Occurrence is generally sporadic, although parent-to-child transmission has been reported on occasion. Employing whole-exome sequencing, we identified heterozygous truncating mutations in SRCAP in five unrelated individuals with sporadic FHS. Sanger sequencing identified mutations in SRCAP in eight more affected persons. Mutations were de novo in all six instances in which parental DNA was available. SRCAP is an SNF2-related chromatin-remodeling factor that serves as a coactivator for CREB-binding protein (CREBBP, better known as CBP, the major cause of Rubinstein-Taybi syndrome [RTS]). Five SRCAP mutations, two of which are recurrent, were identified; all are tightly clustered within a small (111 codon) region of the final exon. These mutations are predicted to abolish three C-terminal AT-hook DNA-binding motifs while leaving the CBP-binding and ATPase domains intact. Our findings show that SRCAP mutations are the major cause of FHS and offer an explanation for the clinical overlap between FHS and RTS.

Refinement of the critical 2p25.3 deletion region: the role of MYT1L in intellectual disability and obesity.

PURPOSE: Submicroscopic deletions of chromosome band 2p25.3 are associated with intellectual disability and/or central obesity. Although MYT1L is believed to be a critical gene responsible for intellectual disability, so far no unequivocal data have confirmed this hypothesis. METHODS: In this study we evaluated a cohort of 22 patients (15 sporadic patients and two families) with a 2p25.3 aberration to further refine the clinical phenotype and to delineate the role of MYT1L in intellectual disability and obesity. In addition, myt1l spatiotemporal expression in zebrafish embryos was analyzed by quantitative polymerase chain reaction and whole-mount in situ hybridization. RESULTS: Complete MYT1L deletion, intragenic deletion, or duplication was observed in all sporadic patients, in addition to two patients with a de novo point mutation in MYT1L. The familial cases comprise a 6-Mb deletion in a father and his three children and a 5' MYT1L overlapping duplication in a father and his two children. Expression analysis in zebrafish embryos shows specific myt1l expression in the developing brain. CONCLUSION: Our data strongly strengthen the hypothesis that MYT1L is the causal gene for the observed syndromal intellectual disability. Moreover, because 17 patients present with obesity/overweight, haploinsufficiency of MYT1L might predispose to weight problems with childhood onset.Genet Med 17 6, 460-466.

Narrative medicine: a call to pens.

An call for papers on the topic of narrative medicine as it pertains to the practice of clinical genetics, this article briefly reviews the tenets of narrative medicine and the ethical issues associated with publishing patients' and doctors' stories. It is an invitation to the readership of the American Journal of Medical Genetics to share its stories of caring for and living with genetic conditions in our new column called Frameshifts.

Trisomy 22 mosaicism and normal developmental outcome: report of two patients and review of the literature.

Mosaic trisomy 22 is known to be compatible with life. However, there are fewer than 20 reports in the literature of live born children and even fewer reports describing their neurodevelopmental outcome. We report on two girls with mosaic trisomy 22 and normal development at ages 7 and 5 years. Both girls had characteristic dysmorphic features including flat nasal bridge, preauricular pits, epicanthic folds, and 5th finger clinodactyly. They also had left-sided hemihyperplasia and short stature. In addition, one of them also had ventricular non-compaction and probable asplenia, two unique features not previously reported. In review of the literature, prenatal and postnatal growth failures were the most common complications of mosaic trisomy 22. Skeletal abnormalities including body asymmetry and 5th finger clinodactyly were also common. While the majority of patients with mosaic trisomy 22 had abnormal cognitive development, normal development has also been documented. It is conceivable that children with trisomy 22 mosaicism, with minimal physical findings and normal development are under diagnosed. Our patients further highlight this potential for normal cognitive outcome and draw attention to possible skewing of unfavorable prognosis for the final developmental outcome in this population. Appropriate information regarding developmental outcome is critical for genetic counseling, especially in prenatal situations.

Duplication of AKT3 as a cause of macrocephaly in duplication 1q43q44.

Somatic and germline duplications of AKT3 and activating mutations of this gene have been reported in individuals with megalencephaly and hemimegalencephaly. We report on a patient with macrocephaly and a 3 Mb duplication on 1q43q44 that includes AKT3. This duplication was detected by array comparative genomic hybridization. The patient presented with moderate developmental delays in gross motor movements and speech. She also had macrocephaly, frontal bossing, hypertelorism, wide nasal bridge, small alae nares, short philtrum, prominent upper lip, and low-set, protruding ears. The 3 Mb duplicated region contained 15 genes including AKT3. The observation of megalencephaly in a child with 1q43q44 duplication provides further evidence of involvement of AKT3 dosage imbalances in brain growth disturbance.

The duplication 17p13.3 phenotype: analysis of 21 families delineates developmental, behavioral and brain abnormalities, and rare variant phenotypes.

Chromosome 17p13.3 is a gene rich region that when deleted is associated with the well-known Miller-Dieker syndrome. A recently described duplication syndrome involving this region has been associated with intellectual impairment, autism and occasional brain MRI abnormalities. We report 34 additional patients from 21 families to further delineate the clinical, neurological, behavioral, and brain imaging findings. We found a highly diverse phenotype with inter- and intrafamilial variability, especially in cognitive development. The most specific phenotype occurred in individuals with large duplications that include both the YWHAE and LIS1 genes. These patients had a relatively distinct facial phenotype and frequent structural brain abnormalities involving the corpus callosum, cerebellar vermis, and cranial base. Autism spectrum disorders were seen in a third of duplication probands, most commonly in those with duplications of YWHAE and flanking genes such as CRK. The typical neurobehavioral phenotype was usually seen in those with the larger duplications. We did not confirm the association of early overgrowth with involvement of YWHAE and CRK, or growth failure with duplications of LIS1. Older patients were often overweight. Three variant phenotypes included cleft lip/palate (CLP), split hand/foot with long bone deficiency (SHFLD), and a connective tissue phenotype resembling Marfan syndrome. The duplications in patients with clefts appear to disrupt ABR, while the SHFLD phenotype was associated with duplication of BHLHA9 as noted in two recent reports. The connective tissue phenotype did not have a convincing critical region. Our experience with this large cohort expands knowledge of this diverse duplication syndrome.

Cornelia de Lange syndrome is caused by mutations in NIPBL, the human homolog of Drosophila melanogaster Nipped-B.

Cornelia de Lange syndrome (CdLS; OMIM 122470) is a dominantly inherited multisystem developmental disorder characterized by growth and cognitive retardation; abnormalities of the upper limbs; gastroesophageal dysfunction; cardiac, ophthalmologic and genitourinary anomalies; hirsutism; and characteristic facial features. Genital anomalies, pyloric stenosis, congenital diaphragmatic hernias, cardiac septal defects, hearing loss and autistic and self-injurious tendencies also frequently occur. Prevalence is estimated to be as high as 1 in 10,000 (ref. 4). We carried out genome-wide linkage exclusion analysis in 12 families with CdLS and identified four candidate regions, of which chromosome 5p13.1 gave the highest multipoint lod score of 2.7. This information, together with the previous identification of a child with CdLS with a de novo t(5;13)(p13.1;q12.1) translocation, allowed delineation of a 1.1-Mb critical region on chromosome 5 for the gene mutated in CdLS. We identified mutations in one gene in this region, which we named NIPBL, in four sporadic and two familial cases of CdLS. We characterized the genomic structure of NIPBL and found that it is widely expressed in fetal and adult tissues. The fly homolog of NIPBL, Nipped-B, facilitates enhancer-promoter communication and regulates Notch signaling and other developmental pathways in Drosophila melanogaster.

Smith-Lemli-Opitz syndrome: phenotype, natural history, and epidemiology.

Smith-Lemli-Opitz syndrome (SLOS) is a congenital multiple anomaly/intellectual disability syndrome caused by a deficiency of cholesterol synthesis resulting from a deficiency of 7-dehydrocholesterol (7DHC) reductase encoded by DHCR7. SLOS is inherited in an autosomal recessive pattern. It is characterized by prenatal and postnatal growth retardation, microcephaly, a variable degree of intellectual disability that encompasses normal intelligence to severe intellectual deficiency, and multiple major and minor malformations. External malformations include distinctive facial features, cleft palate, postaxial polydactyly, 2-3 syndactyly of the toes, and underdeveloped external genitalia in males, while internal anomalies may affect every organ system. The clinical spectrum is wide, and rare individuals have been described with normal development and only minor malformations. The clinical diagnosis of SLOS is confirmed by demonstrating an abnormally elevated concentration of the cholesterol precursor, 7DHC, in serum or other tissues, or by the presence of two DHCR7 mutations. The enzymatic deficiency results in decreased cholesterol and increased 7DHC levels, both during embryonic development and after birth. The malformations found in SLOS may result from decreased cholesterol, increased 7DHC or a combination of these two factors. This review discusses the physical and behavioral phenotype of SLOS, the diagnostic approaches, the natural history from the prenatal period to adulthood, and current understanding of the pathophysiology of SLOS.

Smith-Lemli-Opitz syndrome and other disorders of cholesterol biosynthesis: An introduction.

This issue of Seminars of Medical Genetics features a series of articles on Smith-Lemli-Opitz syndrome and other disorders of endogenous cholesterol synthesis. Clinically, many of these disorders have been known for decades, but only in the last 20 years have the molecular genetic and enzymatic defects underlying these disorders been delineated. As a group, disorders of cholesterol synthesis are relatively common and contribute significantly to the burden of human disease. Leading experts in their fields present clinical, behavioral, molecular, and therapeutic aspects of these disorders.

Novel 9q34.11 gene deletions encompassing combinations of four Mendelian disease genes: STXBP1, SPTAN1, ENG, and TOR1A.

PURPOSE: A number of genes in the 9q34.11 region may be haploinsufficient. However, studies analyzing genotype-phenotype correlations of deletions encompassing multiple dosage-sensitive genes in the region are lacking. METHODS: We mapped breakpoints of 10 patients with 9q34.11 deletions using high-resolution 9q34-specific array comparative genomic hybridization (CGH) to determine deletion size and gene content. RESULTS: The 9q34.11 deletions range in size from 67 kb to 2.8 Mb. Six patients exhibit intellectual disability and share a common deleted region including STXBP1; four manifest variable epilepsy. In five subjects, deletions include SPTAN1, previously associated with early infantile epileptic encephalopathy, infantile spasms, intellectual disability, and hypomyelination. In four patients, the deletion includes endoglin (ENG), causative of hereditary hemorrhagic telangiectasia. Finally, in four patients, deletions involve TOR1A, of which molecular defects lead to early-onset primary dystonia. Ninety-four other RefSeq genes also map to the genomic intervals investigated. CONCLUSION: STXBP1 haploinsufficiency results in progressive encephalopathy characterized by intellectual disability and may be accompanied by epilepsy, movement disorders, and autism. We propose that 9q34.11 genomic deletions involving ENG, TOR1A, STXBP1, and SPTAN1 are responsible for multisystemic vascular dysplasia, early-onset primary dystonia, epilepsy, and intellectual disability, therefore revealing cis-genetic effects leading to complex phenotypes.