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Richard Lifton, Barbara Kazmierczak and colleagues report the identification of a new enterocolitic and autoinflammatory syndrome, which they find is caused by de novo gain-of-function mutations affecting the inflammasome protein NLRC4. Cells with mutant NLRC4 produce elevated levels of cleaved caspase-1, which leads to cell death by pyroptosis. Upon detection of pathogen-associated molecular patterns, innate immune receptors initiate inflammatory responses. These receptors include cytoplasmic NOD-like receptors (NLRs) whose stimulation recruits and proteolytically activates caspase-1 within the inflammasome, a multiprotein complex. Caspase-1 mediates the production of interleukin-1 family cytokines (IL1FCs), leading to fever and inflammatory cell death (pyroptosis) 1 , 2 . Mutations that constitutively activate these pathways underlie several autoinflammatory diseases with diverse clinical features 3 . We describe a family with a previously unreported syndrome featuring neonatal-onset enterocolitis, periodic fever, and fatal or near-fatal episodes of autoinflammation. We show that the disease is caused by a de novo gain-of-function mutation in NLRC4 encoding a p.Val341Ala substitution in the HD1 domain of the protein that cosegregates with disease. Mutant NLRC4 causes constitutive IL1FC production and macrophage cell death. Infected macrophages from affected individuals are polarized toward pyroptosis and exhibit abnormal staining for inflammasome components. These findings identify and describe the cause of a life-threatening but treatable autoinflammatory disease that underscores the divergent roles of the NLRC4 inflammasome.

Non-syndromic craniosynostosis (NSC) is a frequent congenital malformation in which one or more cranial sutures fuse prematurely. Mutations causing rare syndromic craniosynostoses in humans and engineered mouse models commonly increase signaling of the Wnt, bone morphogenetic protein (BMP), or Ras/ERK pathways, converging on shared nuclear targets that promote bone formation. In contrast, the genetics of NSC is largely unexplored. More than 95% of NSC is sporadic, suggesting a role for de novo mutations. Exome sequencing of 291 parent–offspring trios with midline NSC revealed 15 probands with heterozygous damaging de novo mutations in 12 negative regulators of Wnt, BMP, and Ras/ERK signaling (10.9-fold enrichment, P = 2.4 × 10−11). SMAD6 had 4 de novo and 14 transmitted mutations; no other gene had more than 1. Four familial NSC kindreds had mutations in genes previously implicated in syndromic disease. Collectively, these mutations contribute to 10% of probands. Mutations are predominantly loss-of-function, implicating haploinsufficiency as a frequent mechanism. A common risk variant near BMP2 increased the penetrance of SMAD6 mutations and was overtransmitted to patients with de novo mutations in other genes in these pathways, supporting a frequent two-locus pathogenesis. These findings implicate new genes in NSC and demonstrate related pathophysiology of common non-syndromic and rare syndromic craniosynostoses. These findings have implications for diagnosis, risk of recurrence, and risk of adverse neurodevelopmental outcomes. Finally, the use of pathways identified in rare syndromic disease to find genes accounting for non-syndromic cases may prove broadly relevant to understanding other congenital disorders featuring high locus heterogeneity.

Many Mendelian traits are likely unrecognized owing to absence of traditional segregation patterns in families due to causation by de novo mutations, incomplete penetrance, and/or variable expressivity. Genome-level sequencing can overcome these complications. Extreme childhood phenotypes are promising candidates for new Mendelian traits. One example is early onset hypertension, a rare form of a global cause of morbidity and mortality. We performed exome sequencing of 40 unrelated subjects with hypertension due to primary aldosteronism by age 10. Five subjects (12.5%) shared the identical, previously unidentified, heterozygous CACNA1HM1549V mutation. Two mutations were demonstrated to be de novo events, and all mutations occurred independently. CACNA1H encodes a voltage-gated calcium channel (CaV3.2) expressed in adrenal glomerulosa. CACNA1HM1549V showed drastically impaired channel inactivation and activation at more hyperpolarized potentials, producing increased intracellular Ca2+, the signal for aldosterone production. This mutation explains disease pathogenesis and provides new insight into mechanisms mediating aldosterone production and hypertension. The consequence of mutations to the large majority of human genes is unknown. Most mutations that are currently known were discovered by tracing their effects through families. This allows the locations of mutations to be pinpointed on chromosomes—the structures that genetic material is packaged into. Other mutations are harder to trace because individuals with these mutations may develop very different signs and symptoms, or not develop clinical abnormalities at all. Alternatively, a trait may appear sporadically in a family because the mutation arises anew in the affected subject. Recently developed technologies that allow scientists to rapidly sequence all the gene-encoding regions of an individual's DNA—their genome—offer a new way to identify harmful genetic variants. Comparing the genomes of individuals with rare disorders can reveal if the individuals share any genetic mutations in common that could cause their symptoms. Scholl et al. used this strategy to sequence the genomes of 40 individuals with a rare type of hypertension—a condition that causes high blood pressure, and increases the risk of strokes, kidney failure and heart attacks—that develops early in childhood. In this form of the disease, high blood pressure is caused by the adrenal glands above the kidneys producing too much of a hormone called aldosterone. Some genetic causes of this form of the disease have already been identified. Now, Scholl et al. have found a new genetic mutation present in five families with this condition. Two of the individuals were the first in their families to develop this mutation, while three others inherited it. Some of the family members with this mutation had hypertension and some did not. The mutation is in a gene that encodes a type of calcium channel—a protein found in the membrane that surrounds cells, and which can open and close to control the amount of calcium in the cell. This particular calcium channel is abundant in the cells of the adrenal gland. Scholl et al. found that the mutation causes the calcium channels to be more likely to open and take longer to close. This increases the number of calcium ions that move into the cell, which causes the adrenal gland to produce more aldosterone. These new insights have provided a new way of diagnosing early-onset hypertension, and suggest that targeting calcium channels could help to develop new treatments for this disease.

Premature fusion of the cranial sutures (craniosynostosis), affecting 1 in 2000 newborns, is treated surgically in infancy to prevent adverse neurologic outcomes. To identify mutations contributing to common non-syndromic midline (sagittal and metopic) craniosynostosis, we performed exome sequencing of 132 parent-offspring trios and 59 additional probands. Thirteen probands (7%) had damaging de novo or rare transmitted mutations in SMAD6, an inhibitor of BMP – induced osteoblast differentiation (p<10−20). SMAD6 mutations nonetheless showed striking incomplete penetrance (<60%). Genotypes of a common variant near BMP2 that is strongly associated with midline craniosynostosis explained nearly all the phenotypic variation in these kindreds, with highly significant evidence of genetic interaction between these loci via both association and analysis of linkage. This epistatic interaction of rare and common variants defines the most frequent cause of midline craniosynostosis and has implications for the genetic basis of other diseases. The bones in the front, back and sides of the human skull are not fused to one another at birth in order to allow the brain to double in size during the first year of life and continue growing into adulthood. However, one in 2,000 infants is born with a condition called craniosynostosis in which some of these bones have already fused. This fusion prevents the skull from growing properly, and can lead to the brain becoming compressed. As such, surgeons routinely undo the fusion in these infants to allow the brain and skull to grow normally. Eighty-five percent of craniosynostosis cases occur in infants with no other abnormalities, (called non-syndromic cases) and most have no other affected family member. It has therefore been unclear whether these infants have craniosynostosis due to a genetic or non-genetic cause. If the cause is genetic, it is also not clear whether a mutation in a single gene, the combined effects of many genes, or something in between is responsible. Now, by focusing on a group of 191 infants with premature fusion of bones joined at the midline of the skull, Timberlake et al. asked if any of the approximately 20,000 genes in the human genome were altered more frequently in these infants than would be expected by chance. This search revealed that rare mutations that disable one copy of a gene called SMAD6 in combination with a common DNA variant near another gene called BMP2 account for about 7% of infants with midline forms of craniosynostosis. These genes are both known to regulate how bones form, which explains how the mutation of these genes could lead to craniosynostosis. In all cases, the parents of these children were unaffected. This was typically because one parent had only the SMAD6 mutation while the other had only the common BMP2 variant; the transmission of both to their offspring resulted in craniosynostosis. The finding that a rare mutation’s effect is strongly modified by a common variant from another site in the genome is unprecedented. These findings will allow doctors to counsel families about the risk of having additional children with craniosynostosis. Timberlake et al. next plan to study more patients with craniosynostosis to identify additional genes that contribute to this disease. They will also look at other diseases to see whether the combination of rare mutation and common DNA variant could be behind other unexplained disorders.

Genetic investigation of inherited skin disorders has informed the understanding of skin self-renewal, differentiation, and barrier function. Erythrokeratodermia variabilis et progressiva (EKVP) is a rare, inherited skin disease that is characterized by transient figurate patches of erythema, localized or generalized scaling, and frequent palmoplantar keratoderma. By using exome sequencing, we show that de novo missense mutations in GJA1 (gap junction protein alpha 1) cause EKVP. The severe, progressive skin disease in EKVP subjects with GJA1 mutations is distinct from limited cutaneous findings rarely found in the systemic disorder oculodentodigital dysplasia, also caused by dominant GJA1 mutations. GJA1 encodes connexin 43 (Cx43), the most widely expressed gap junction protein. We show that the GJA1 mutations in EKVP subjects lead to disruption of Cx43 membrane localization and aggregation within the Golgi. These findings reveal a critical role for Cx43 in epidermal homeostasis, and they provide evidence of organ-specific pathobiology resulting from different mutations within GJA1.

Primary aldosteronism, a common cause of severe hypertension 1 , features constitutive production of the adrenal steroid aldosterone. We analyzed a multiplex family with familial hyperaldosteronism type II (FH-II) 2 and 80 additional probands with unsolved early-onset primary aldosteronism. Eight probands had novel heterozygous variants in CLCN2 , including two de novo mutations and four independent occurrences of a mutation encoding an identical p.Arg172Gln substitution; all relatives with early-onset primary aldosteronism carried the CLCN2 variant found in the proband. CLCN2 encodes a voltage-gated chloride channel expressed in adrenal glomerulosa that opens at hyperpolarized membrane potentials. Channel opening depolarizes glomerulosa cells and induces expression of aldosterone synthase, the rate-limiting enzyme for aldosterone biosynthesis. Mutant channels show gain of function, with higher open probabilities at the glomerulosa resting potential. These findings for the first time demonstrate a role of anion channels in glomerulosa membrane potential determination, aldosterone production and hypertension. They establish the cause of a substantial fraction of early-onset primary aldosteronism. Whole-exome sequencing identifies mutations in CLCN2 in individuals with familial hyperaldosteronism type II or early-onset primary aldosteronism. These gain-of-function mutations cause chloride channel opening and glomerulosa cell depolarization, showing a role for anion channels in aldosterone production.

Richard Lifton and colleagues identify somatic and germline mutations in the CACNA1D calcium channel gene in aldosterone-producing adenomas and primary aldosteronism. Their functional studies show that these mutations result in channel activation at more hyperpolarized membrane potentials, implicating increased Ca 2+ influx in disease pathogenesis. Adrenal aldosterone-producing adenomas (APAs) constitutively produce the salt-retaining hormone aldosterone and are a common cause of severe hypertension. Recurrent mutations in the potassium channel gene KCNJ5 that result in cell depolarization and Ca 2+ influx cause ∼40% of these tumors 1 . We identified 5 somatic mutations (4 altering Gly403 and 1 altering Ile770) in CACNA1D , encoding a voltage-gated calcium channel, among 43 APAs without mutated KCNJ5 . The altered residues lie in the S6 segments that line the channel pore. Both alterations result in channel activation at less depolarized potentials; Gly403 alterations also impair channel inactivation. These effects are inferred to cause increased Ca 2+ influx, which is a sufficient stimulus for aldosterone production and cell proliferation in adrenal glomerulosa 2 . We also identified de novo germline mutations at identical positions in two children with a previously undescribed syndrome featuring primary aldosteronism and neuromuscular abnormalities. These findings implicate gain-of-function Ca 2+ channel mutations in APAs and primary aldosteronism.

Lambdoid craniosynostosis (CS) is a congenital anomaly resulting from premature fusion of the cranial suture between the parietal and occipital bones. Predominantly sporadic, it is the rarest form of CS and its genetic etiology is largely unexplored. Exome sequencing of 25 kindreds, including 18 parent–offspring trios with sporadic lambdoid CS, revealed a marked excess of damaging (predominantly missense) de novo mutations that account for ~ 40% of sporadic cases. These mutations clustered in the BMP signaling cascade (P = 1.6 × 10–7), including mutations in genes encoding BMP receptors (ACVRL1 and ACVR2A), transcription factors (SOX11, FOXO1) and a transcriptional co-repressor (IFRD1), none of which have been implicated in other forms of CS. These missense mutations are at residues critical for substrate or target sequence recognition and many are inferred to cause genetic gain-of-function. Additionally, mutations in transcription factor NFIX were implicated in syndromic craniosynostosis affecting diverse sutures. Single cell RNA sequencing analysis of the mouse lambdoid suture identified enrichment of mutations in osteoblast precursors (P = 1.6 × 10−6), implicating perturbations in the balance between proliferation and differentiation of osteoprogenitor cells in lambdoid CS. The results contribute to the growing knowledge of the genetics of CS, have implications for genetic counseling, and further elucidate the molecular etiology of premature suture fusion.