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PRKACA and PRKACB code for two catalytic subunits (Cα and Cβ) of cAMP-dependent protein kinase (PKA), a pleiotropic holoenzyme that regulates numerous fundamental biological processes such as metabolism, development, memory, and immune response. We report seven unrelated individuals presenting with a multiple congenital malformation syndrome in whom we identified heterozygous germline or mosaic missense variants in PRKACA or PRKACB. Three affected individuals were found with the same PRKACA variant, and the other four had different PRKACB mutations. In most cases, the mutations arose de novo, and two individuals had offspring with the same condition. Nearly all affected individuals and their affected offspring shared an atrioventricular septal defect or a common atrium along with postaxial polydactyly. Additional features included skeletal abnormalities and ectodermal defects of variable severity in five individuals, cognitive deficit in two individuals, and various unusual tumors in one individual. We investigated the structural and functional consequences of the variants identified in PRKACA and PRKACB through the use of several computational and experimental approaches, and we found that they lead to PKA holoenzymes which are more sensitive to activation by cAMP than are the wild-type proteins. Furthermore, expression of PRKACA or PRKACB variants detected in the affected individuals inhibited hedgehog signaling in NIH 3T3 fibroblasts, thereby providing an underlying mechanism for the developmental defects observed in these cases. Our findings highlight the importance of both Cα and Cβ subunits of PKA during human development., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Glypicans are a family of cell-surface heparan sulfate proteoglycans that regulate growth-factor signaling during development and are thought to play a role in the regulation of morphogenesis. Whole-exome sequencing of the Australian family that defined Keipert syndrome (nasodigitoacoustic syndrome) identified a hemizygous truncating variant in the gene encoding glypican 4 (GPC4). This variant, located in the final exon of GPC4, results in premature termination of the protein 51 amino acid residues prior to the stop codon, and in concomitant loss of functionally important N-linked glycosylation (Asn514) and glycosylphosphatidylinositol (GPI) anchor (Ser529) sites. We subsequently identified seven affected males from five additional kindreds with novel and predicted pathogenic variants in GPC4. Segregation analysis and X-inactivation studies in carrier females provided supportive evidence that the GPC4 variants caused the condition. Furthermore, functional studies of recombinant protein suggested that the truncated proteins p.Gln506 ∗ and p.Glu496 ∗ were less stable than the wild type. Clinical features of Keipert syndrome included a prominent forehead, a flat midface, hypertelorism, a broad nose, downturned corners of mouth, and digital abnormalities, whereas cognitive impairment and deafness were variable features. Studies of Gpc4 knockout mice showed evidence of the two primary features of Keipert syndrome: craniofacial abnormalities and digital abnormalities. Phylogenetic analysis demonstrated that GPC4 is most closely related to GPC6, which is associated with a bone dysplasia that has a phenotypic overlap with Keipert syndrome. Overall, we have shown that pathogenic variants in GPC4 cause a loss of function that results in Keipert syndrome, making GPC4 the third human glypican to be linked to a genetic syndrome., (Crown Copyright © 2019. Published by Elsevier Inc. All rights reserved.)

Neurofibromatosis type 1 (NF1), a common genetic disorder with a birth incidence of 1:2,000-3,000, is characterized by a highly variable clinical presentation. To date, only two clinically relevant intragenic genotype-phenotype correlations have been reported for NF1 missense mutations affecting p.Arg1809 and a single amino acid deletion p.Met922del. Both variants predispose to a distinct mild NF1 phenotype with neither externally visible cutaneous/plexiform neurofibromas nor other tumors. Here, we report 162 individuals (129 unrelated probands and 33 affected relatives) heterozygous for a constitutional missense mutation affecting one of five neighboring NF1 codons-Leu844, Cys845, Ala846, Leu847, and Gly848-located in the cysteine-serine-rich domain (CSRD). Collectively, these recurrent missense mutations affect ∼0.8% of unrelated NF1 mutation-positive probands in the University of Alabama at Birmingham (UAB) cohort. Major superficial plexiform neurofibromas and symptomatic spinal neurofibromas were more prevalent in these individuals compared with classic NF1-affected cohorts (both p < 0.0001). Nearly half of the individuals had symptomatic or asymptomatic optic pathway gliomas and/or skeletal abnormalities. Additionally, variants in this region seem to confer a high predisposition to develop malignancies compared with the general NF1-affected population (p = 0.0061). Our results demonstrate that these NF1 missense mutations, although located outside the GAP-related domain, may be an important risk factor for a severe presentation. A genotype-phenotype correlation at the NF1 region 844-848 exists and will be valuable in the management and genetic counseling of a significant number of individuals., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Autosomal-dominant tubulo-interstitial kidney disease (ADTKD) encompasses a group of disorders characterized by renal tubular and interstitial abnormalities, leading to slow progressive loss of kidney function requiring dialysis and kidney transplantation. Mutations in UMOD, MUC1, and REN are responsible for many, but not all, cases of ADTKD. We report on two families with ADTKD and congenital anemia accompanied by either intrauterine growth retardation or neutropenia. Ultrasound and kidney biopsy revealed small dysplastic kidneys with cysts and tubular atrophy with secondary glomerular sclerosis, respectively. Exclusion of known ADTKD genes coupled with linkage analysis, whole-exome sequencing, and targeted re-sequencing identified heterozygous missense variants in SEC61A1-c.553A>G (p.Thr185Ala) and c.200T>G (p.Val67Gly)-both affecting functionally important and conserved residues in SEC61. Both transiently expressed SEC6A1A variants are delocalized to the Golgi, a finding confirmed in a renal biopsy from an affected individual. Suppression or CRISPR-mediated deletions of sec61al2 in zebrafish embryos induced convolution defects of the pronephric tubules but not the pronephric ducts, consistent with the tubular atrophy observed in the affected individuals. Human mRNA encoding either of the two pathogenic alleles failed to rescue this phenotype as opposed to a complete rescue by human wild-type mRNA. Taken together, these findings provide a mechanism by which mutations in SEC61A1 lead to an autosomal-dominant syndromic form of progressive chronic kidney disease. We highlight protein translocation defects across the endoplasmic reticulum membrane, the principal role of the SEC61 complex, as a contributory pathogenic mechanism for ADTKD., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Desbuquois dysplasia (DBQD) is a severe condition characterized by short stature, joint laxity, and advanced carpal ossification. Based on the presence of additional hand anomalies, we have previously distinguished DBQD type 1 and identified CANT1 (calcium activated nucleotidase 1) mutations as responsible for DBQD type 1. We report here the identification of five distinct homozygous xylosyltransferase 1 (XYLT1) mutations in seven DBQD type 2 subjects from six consanguineous families. Among the five mutations, four were expected to result in loss of function and a drastic reduction of XYLT1 cDNA level was demonstrated in two cultured individual fibroblasts. Because xylosyltransferase 1 (XT-I) catalyzes the very first step in proteoglycan (PG) biosynthesis, we further demonstrated in the two individual fibroblasts a significant reduction of cellular PG content. Our findings of XYLT1 mutations in DBQD type 2 further support a common physiological basis involving PG synthesis in the multiple dislocation group of disorders. This observation sheds light on the key role of the XT-I during the ossification process., (Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Genomic rearrangements involving AUTS2 (7q11.22) are associated with autism and intellectual disability (ID), although evidence for causality is limited. By combining the results of diagnostic testing of 49,684 individuals, we identified 24 microdeletions that affect at least one exon of AUTS2, as well as one translocation and one inversion each with a breakpoint within the AUTS2 locus. Comparison of 17 well-characterized individuals enabled identification of a variable syndromic phenotype including ID, autism, short stature, microcephaly, cerebral palsy, and facial dysmorphisms. The dysmorphic features were more pronounced in persons with 3'AUTS2 deletions. This part of the gene is shown to encode a C-terminal isoform (with an alternative transcription start site) expressed in the human brain. Consistent with our genetic data, suppression of auts2 in zebrafish embryos caused microcephaly that could be rescued by either the full-length or the C-terminal isoform of AUTS2. Our observations demonstrate a causal role of AUTS2 in neurocognitive disorders, establish a hitherto unappreciated syndromic phenotype at this locus, and show how transcriptional complexity can underpin human pathology. The zebrafish model provides a valuable tool for investigating the etiology of AUTS2 syndrome and facilitating gene-function analysis in the future., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Spondyloepimetaphyseal dysplasia with joint laxity, leptodactylic type (lepto-SEMDJL, aka SEMDJL, Hall type), is an autosomal dominant skeletal disorder that, in spite of being relatively common among skeletal dysplasias, has eluded molecular elucidation so far. We used whole-exome sequencing of five unrelated individuals with lepto-SEMDJL to identify mutations in KIF22 as the cause of this skeletal condition. Missense mutations affecting one of two adjacent amino acids in the motor domain of KIF22 were present in 20 familial cases from eight families and in 12 other sporadic cases. The skeletal and connective tissue phenotype produced by these specific mutations point to functions of KIF22 beyond those previously ascribed functions involving chromosome segregation. Although we have found Kif22 to be strongly upregulated at the growth plate, the precise pathogenetic mechanisms remain to be elucidated., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Geleophysic (GD) and acromicric dysplasia (AD) belong to the acromelic dysplasia group and are both characterized by severe short stature, short extremities, and stiff joints. Although AD has an unknown molecular basis, we have previously identified ADAMTSL2 mutations in a subset of GD patients. After exome sequencing in GD and AD cases, we selected fibrillin 1 (FBN1) as a candidate gene, even though mutations in this gene have been described in Marfan syndrome, which is characterized by tall stature and arachnodactyly. We identified 16 heterozygous FBN1 mutations that are all located in exons 41 and 42 and encode TGFβ-binding protein-like domain 5 (TB5) of FBN1 in 29 GD and AD cases. Microfibrillar network disorganization and enhanced TGFβ signaling were consistent features in GD and AD fibroblasts. Importantly, a direct interaction between ADAMTSL2 and FBN1 was demonstrated, suggesting a disruption of this interaction as the underlying mechanism of GD and AD phenotypes. Although enhanced TGFβ signaling caused by FBN1 mutations can trigger either Marfan syndrome or GD and AD, our findings support the fact that TB5 mutations in FBN1 are responsible for short stature phenotypes., (Copyright © 2011 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

By using a combination of array comparative genomic hybridization and a candidate gene approach, we identified nuclear factor I/X (NFIX) deletions or nonsense mutation in three sporadic cases of a Sotos-like overgrowth syndrome with advanced bone age, macrocephaly, developmental delay, scoliosis, and unusual facies. Unlike the aforementioned human syndrome, Nfix-deficient mice are unable to gain weight and die in the first 3 postnatal weeks, while they also present with a spinal deformation and decreased bone mineralization. These features prompted us to consider NFIX as a candidate gene for Marshall-Smith syndrome (MSS), a severe malformation syndrome characterized by failure to thrive, respiratory insufficiency, accelerated osseous maturation, kyphoscoliosis, osteopenia, and unusual facies. Distinct frameshift and splice NFIX mutations that escaped nonsense-mediated mRNA decay (NMD) were identified in nine MSS subjects. NFIX belongs to the Nuclear factor one (NFI) family of transcription factors, but its specific function is presently unknown. We demonstrate that NFIX is normally expressed prenatally during human brain development and skeletogenesis. These findings demonstrate that allelic NFIX mutations trigger distinct phenotypes, depending specifically on their impact on NMD.

Congenital heart defects (CHDs) are the most common major developmental anomalies and the most frequent cause for perinatal mortality, but their etiology remains often obscure. We identified a locus for CHDs on 6q24-q25. Genotype-phenotype correlations in 12 patients carrying a chromosomal deletion on 6q delineated a critical 850 kb region on 6q25.1 harboring five genes. Bioinformatics prioritization of candidate genes in this locus for a role in CHDs identified the TGF-beta-activated kinase 1/MAP3K7 binding protein 2 gene (TAB2) as the top-ranking candidate gene. A role for this candidate gene in cardiac development was further supported by its conserved expression in the developing human and zebrafish heart. Moreover, a critical, dosage-sensitive role during development was demonstrated by the cardiac defects observed upon titrated knockdown of tab2 expression in zebrafish embryos. To definitively confirm the role of this candidate gene in CHDs, we performed mutation analysis of TAB2 in 402 patients with a CHD, which revealed two evolutionarily conserved missense mutations. Finally, a balanced translocation was identified, cosegregating with familial CHD. Mapping of the breakpoints demonstrated that this translocation disrupts TAB2. Taken together, these data clearly demonstrate a role for TAB2 in human cardiac development., (Copyright 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Spondylo-megaepiphyseal-metaphyseal dysplasia (SMMD) is a rare skeletal dysplasia with only a few cases reported in the literature. Affected individuals have a disproportionate short stature with a short and stiff neck and trunk. The limbs appear relatively long and may show flexion contractures of the distal joints. The most remarkable radiographic features are the delayed and impaired ossification of the vertebral bodies as well as the presence of large epiphyseal ossification centers and wide growth plates in the long tubular bones. Numerous pseudoepiphyses of the short tubular bones in hands and feet are another remarkable feature of the disorder. Genome wide homozygosity mapping followed by a candidate gene approach resulted in the elucidation of the genetic cause in three new consanguineous families with SMMD. Each proband was homozygous for a different inactivating mutation in NKX3-2, a homeobox-containing gene located on chromosome 4p15.33. Striking similarities were found when comparing the vertebral ossification defects in SMMD patients with those observed in the Nkx3-2 null mice. Distinguishing features were the asplenia found in the mutant mice and the radiographic abnormalities in the limbs only observed in SMMD patients. The absence of the latter anomalies in the murine model may be due to the perinatal death of the affected animals. This study illustrates that NKX3-2 plays an important role in endochondral ossification of both the axial and appendicular skeleton in humans. In addition, it defines SMMD as yet another skeletal dysplasia with autosomal-recessive inheritance and a distinct phenotype.

Desbuquois dysplasia is a severe condition characterized by short stature, joint laxity, scoliosis, and advanced carpal ossification with a delta phalanx. Studying nine Desbuquois families, we identified seven distinct mutations in the Calcium-Activated Nucleotidase 1 gene (CANT1), which encodes a soluble UDP-preferring nucleotidase belonging to the apyrase family. Among the seven mutations, four were nonsense mutations (Del 5' UTR and exon 1, p.P245RfsX3, p.S303AfsX20, and p.W125X), and three were missense mutations (p.R300C, p.R300H, and p.P299L) responsible for the change of conserved amino acids located in the seventh nucleotidase conserved region (NRC). The arginine substitution at position 300 was identified in five out of nine families. The specific function of CANT1 is as yet unknown, but its substrates are involved in several major signaling functions, including Ca2+ release, through activation of pyrimidinergic signaling. Importantly, using RT-PCR analysis, we observed a specific expression in chondrocytes. We also found electron-dense material within distended rough endoplasmic reticulum in the fibroblasts of Desbuquois patients. Our findings demonstrate the specific involvement of a nucleotidase in the endochondral ossification process.

Mutations in the RMRP gene lead to a wide spectrum of autosomal recessive skeletal dysplasias, ranging from the milder phenotypes metaphyseal dysplasia without hypotrichosis and cartilage hair hypoplasia (CHH) to the severe anauxetic dysplasia (AD). This clinical spectrum includes different degrees of short stature, hair hypoplasia, defective erythrogenesis, and immunodeficiency. The RMRP gene encodes the untranslated RNA component of the mitochondrial RNA-processing ribonuclease, RNase MRP. We recently demonstrated that mutations may affect both messenger RNA (mRNA) and ribosomal RNA (rRNA) cleavage and thus cell-cycle regulation and protein synthesis. To investigate the genotype-phenotype correlation, we analyzed the position and the functional effect of 13 mutations in patients with variable features of the CHH-AD spectrum. Those at the end of the spectrum include a novel patient with anauxetic dysplasia who was compound heterozygous for the null mutation g.254&#95;263delCTCAGCGCGG and the mutation g.195C-->T, which was previously described in patients with milder phenotypes. Mapping of nucleotide conservation to the two-dimensional structure of the RMRP gene revealed that disease-causing mutations either affect evolutionarily conserved nucleotides or are likely to alter secondary structure through mispairing in stem regions. In vitro testing of RNase MRP multiprotein-specific mRNA and rRNA cleavage of different mutations revealed a strong correlation between the decrease in rRNA cleavage in ribosomal assembly and the degree of bone dysplasia, whereas reduced mRNA cleavage, and thus cell-cycle impairment, predicts the presence of hair hypoplasia, immunodeficiency, and hematological abnormalities and thus increased cancer risk.

We observed two unrelated consanguineous families with malformation syndromes sharing anophthalmia and distinct eyebrows as common signs, but differing for alveolar capillary dysplasia or complex congenital heart defect in one and diaphragmatic hernia in the other family. Homozygosity mapping revealed linkage to a common locus on chromosome 15, and pathogenic homozygous mutations were identified in STRA6, a member of a large group of "stimulated by retinoic acid" genes encoding novel transmembrane proteins, transcription factors, and secreted signaling molecules or proteins of largely unknown function. Subsequently, homozygous STRA6 mutations were also demonstrated in 3 of 13 patients chosen on the basis of significant phenotypic overlap to the original cases. While a homozygous deletion generating a premature stop codon (p.G50AfsX22) led to absence of the immunoreactive protein in patient's fibroblast culture, structural analysis of three missense mutations (P90L, P293L, and T321P) suggested significant effects on the geometry of the loops connecting the transmembrane helices of STRA6. Two further variations in the C-terminus (T644M and R655C) alter specific functional sites, an SH2-binding motif and a phosphorylation site, respectively. STRA6 mutations thus define a pleiotropic malformation syndrome representing the first human phenotype associated with mutations in a gene from the "STRA" group.

Multiple epiphyseal dysplasia (MED) is a genetically heterogeneous disorder with marked clinical and radiographic variability. Traditionally, the mild "Ribbing" and severe "Fairbank" types have been used to define a broad phenotypic spectrum. Mutations in the gene encoding cartilage oligomeric-matrix protein have been shown to result in several types of MED, whereas mutations in the gene encoding the alpha2 chain of type IX collagen (COL9A2) have so far been found only in two families with the Fairbank type of MED. Type IX collagen is a heterotrimer of pro-alpha chains derived from three distinct genes-COL9A1, COL9A2, and COL9A3. In this article, we describe two families with distinctive oligo-epiphyseal forms of MED, which are heterozygous for different mutations in the COL9A2 exon 3/intron 3 splice-donor site. Both of these mutations result in the skipping of exon 3 from COL9A2 mRNA, but the position of the mutation in the splice-donor site determines the stability of the mRNA produced from the mutant COL9A2 allele.

Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (MED) are autosomal dominant osteochondrodysplasias that result in mild to severe short-limb dwarfism and early-onset osteoarthrosis. PSACH and some forms of MED result from mutations in the gene for cartilage oligomeric matrix protein (COMP; OMIM 600310 [http://www3.ncbi.nlm. nih.gov:80/htbin-post/Omim/dispmim?600310]). We report the identification of COMP mutations in an additional 14 families with PSACH or MED phenotypes. Mutations predicted to result in single-amino acid deletions or substitutions, all in the region of the COMP gene encoding the calmodulin-like repeat elements, were identified in patients with moderate to severe PSACH. We also identified within this domain a missense mutation that produced MED Fairbank. In two families, one with mild PSACH and the second with a form of MED, we identified different substitutions for a residue in the carboxyl-terminal globular region of COMP. Both the clinical presentations of these two families and the identification of COMP-gene mutations provide evidence of phenotypic overlap between PSACH and MED. These data also reveal a role for the carboxyl-terminal domain in the structure and/or function of COMP.

Hereditary multiple exostoses (EXT; MIM 133700) is an autosomal dominant bone disorder characterized by the presence of multiple benign cartilage-capped tumors (exostoses). Besides suffering complications caused by the pressure of these exostoses on the surrounding tissues, EXT patients are at an increased risk for malignant chondrosarcoma, which may develop from an exostosis. EXT is genetically heterogeneous, and three loci have been identified so far: EXT1, on chromosome 8q23-q24; EXT2, on 11p11-p12; and EXT3, on the short arm of chromosome 19. The EXT1 and EXT2 genes were cloned recently, and they were shown to be homologous. We have now analyzed the EXT1 and EXT2 genes, in 26 EXT families originating from nine countries, to identify the underlying disease-causing mutation. Of the 26 families, 10 families had an EXT1 mutation, and 10 had an EXT2 mutation. Twelve of these mutations have never been described before. In addition, we have reviewed all EXT1 and EXT2 mutations reported so far, to determine the nature, frequency, and distribution of mutations that cause EXT. From this analysis, we conclude that mutations in either the EXT1 or the EXT2 gene are responsible for the majority of EXT cases. Most of the mutations in EXT1 and EXT2 cause premature termination of the EXT proteins, whereas missense mutations are rare. The development is thus mainly due to loss of function of the EXT genes, consistent with the hypothesis that the EXT genes have a tumor- suppressor function.