IFT74 variants cause skeletal ciliopathy and motile cilia defects in mice and humans.

Motile and non-motile cilia play critical roles in mammalian development and health. These organelles are composed of a 1000 or more unique proteins, but their assembly depends entirely on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). In mammals, malfunction of non-motile cilia due to IFT dysfunction results in complex developmental phenotypes that affect most organs. In contrast, disruption of motile cilia function causes subfertility, disruption of the left-right body axis, and recurrent airway infections with progressive lung damage. In this work, we characterize allele specific phenotypes resulting from IFT74 dysfunction in human and mice. We identified two families carrying a deletion encompassing IFT74 exon 2, the first coding exon, resulting in a protein lacking the first 40 amino acids and two individuals carrying biallelic splice site mutations. Homozygous exon 2 deletion cases presented a ciliary chondrodysplasia with narrow thorax and progressive growth retardation along with a mucociliary clearance disorder phenotype with severely shorted cilia. Splice site variants resulted in a lethal skeletal chondrodysplasia phenotype. In mice, removal of the first 40 amino acids likewise results in a motile cilia phenotype but with little effect on primary cilia structure. Mice carrying this allele are born alive but are growth restricted and developed hydrocephaly in the first month of life. In contrast, a strong, likely null, allele of Ift74 in mouse completely blocks ciliary assembly and causes severe heart defects and midgestational lethality. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia resulting from increased mechanical stress and repair needs could account for the motile cilia phenotype observed in human and mice.

Associations of height, body mass index, and weight gain with breast cancer risk in carriers of a pathogenic variant in BRCA1 or BRCA2: the BRCA1 and BRCA2 Cohort Consortium.

INTRODUCTION: Height, body mass index (BMI), and weight gain are associated with breast cancer risk in the general population. It is unclear whether these associations also exist for carriers of pathogenic variants in the BRCA1 or BRCA2 genes. PATIENTS AND METHODS: An international pooled cohort of 8091 BRCA1/2 variant carriers was used for retrospective and prospective analyses separately for premenopausal and postmenopausal women. Cox regression was used to estimate breast cancer risk associations with height, BMI, and weight change. RESULTS: In the retrospective analysis, taller height was associated with risk of premenopausal breast cancer for BRCA2 variant carriers (HR 1.20 per 10 cm increase, 95% CI 1.04-1.38). Higher young-adult BMI was associated with lower premenopausal breast cancer risk for both BRCA1 (HR 0.75 per 5 kg/m2, 95% CI 0.66-0.84) and BRCA2 (HR 0.76, 95% CI 0.65-0.89) variant carriers in the retrospective analysis, with consistent, though not statistically significant, findings from the prospective analysis. In the prospective analysis, higher BMI and adult weight gain were associated with higher postmenopausal breast cancer risk for BRCA1 carriers (HR 1.20 per 5 kg/m2, 95% CI 1.02-1.42; and HR 1.10 per 5 kg weight gain, 95% CI 1.01-1.19, respectively). CONCLUSION: Anthropometric measures are associated with breast cancer risk for BRCA1 and BRCA2 variant carriers, with relative risk estimates that are generally consistent with those for women from the general population.

IFT74 variants cause skeletal ciliopathy and motile cilia defects in mice and humans.

Motile and non-motile cilia are critical to mammalian development and health. Assembly of these organelles depends on proteins synthesized in the cell body and transported into the cilium by intraflagellar transport (IFT). A series of human and mouse IFT74 variants were studied to understand the function of this IFT subunit. Humans missing exon 2, which codes for the first 40 residues, presented an unusual combination of ciliary chondrodysplasia and mucociliary clearance disorders while individuals carrying biallelic splice site variants developed a lethal skeletal chondrodysplasia. In mice, variants thought to remove all Ift74 function, completely block ciliary assembly and result in midgestational lethality. A mouse allele that removes the first 40 amino acids, analogous to the human exon 2 deletion, results in a motile cilia phenotype with mild skeletal abnormalities. In vitro studies suggest that the first 40 amino acids of IFT74 are dispensable for binding of other IFT subunits but are important for tubulin binding. Higher demands on tubulin transport in motile cilia compared to primary cilia could account for the motile cilia phenotype observed in human and mice.

Comprehensive study of 28 individuals with SIN3A-related disorder underscoring the associated mild cognitive and distinctive facial phenotype.

Witteveen-Kolk syndrome (OMIM 613406) is a recently defined neurodevelopmental syndrome caused by heterozygous loss-of-function variants in SIN3A. We define the clinical and neurodevelopmental phenotypes related to SIN3A-haploinsufficiency in 28 unreported patients. Patients with SIN3A variants adversely affecting protein function have mild intellectual disability, growth and feeding difficulties. Involvement of a multidisciplinary team including a geneticist, paediatrician and neurologist should be considered in managing these patients. Patients described here were identified through a combination of clinical evaluation and gene matching strategies (GeneMatcher and Decipher). All patients consented to participate in this study. Mean age of this cohort was 8.2 years (17 males, 11 females). Out of 16 patients ≥ 8 years old assessed, eight (50%) had mild intellectual disability (ID), four had moderate ID (22%), and one had severe ID (6%). Four (25%) did not have any cognitive impairment. Other neurological symptoms such as seizures (4/28) and hypotonia (12/28) were common. Behaviour problems were reported in a minority. In patients ≥2 years, three were diagnosed with Autism Spectrum Disorder (ASD) and four with Attention Deficit Hyperactivity Disorder (ADHD). We report 27 novel variants and one previously reported variant. 24 were truncating variants; three were missense variants and one large in-frame gain including exons 10-12.

Refining the Primrose syndrome phenotype: A study of five patients with ZBTB20 de novo variants and a review of the literature.

Primrose syndrome is a rare autosomal dominant condition caused by heterozygous missense variants within ZBTB20. Through an exome sequencing approach (as part of the Deciphering Developmental Disorders [DDD] study) we have identified five unrelated individuals with previously unreported, de novo ZBTB20 pathogenic missense variants. All five missense variants targeted the C2H2 zinc finger domains. This genotype-up approach has allowed further refinement of the Primrose syndrome phenotype. Major characteristics (>90% individuals) include an intellectual disability (most frequently in the moderate range), a recognizable facial appearance and brain MRI abnormalities, particularly abnormalities of the corpus callosum. Other frequent clinical associations (in 50-90% individuals) include sensorineural hearing loss (83%), hypotonia (78%), cryptorchidism in males (75%), macrocephaly (72%), behavioral issues (56%), and dysplastic/hypoplastic nails (57%). Based upon these clinical data we discuss our current management of patients with Primrose syndrome.

Congenital hypogonadotropic hypogonadism and constitutional delay of growth and puberty have distinct genetic architectures.

OBJECTIVE: Congenital hypogonadotropic hypogonadism (CHH) and constitutional delay of growth and puberty (CDGP) represent rare and common forms of GnRH deficiency, respectively. Both CDGP and CHH present with delayed puberty, and the distinction between these two entities during early adolescence is challenging. More than 30 genes have been implicated in CHH, while the genetic basis of CDGP is poorly understood. DESIGN: We characterized and compared the genetic architectures of CHH and CDGP, to test the hypothesis of a shared genetic basis between these disorders. METHODS: Exome sequencing data were used to identify rare variants in known genes in CHH (n = 116), CDGP (n = 72) and control cohorts (n = 36 874 ExAC and n = 405 CoLaus). RESULTS: Mutations in at least one CHH gene were found in 51% of CHH probands, which is significantly higher than in CDGP (7%, P = 7.6 × 10-11) or controls (18%, P = 5.5 × 10-12). Similarly, oligogenicity (defined as mutations in more than one gene) was common in CHH patients (15%) relative to CDGP (1.4%, P = 0.002) and controls (2%, P = 6.4 × 10-7). CONCLUSIONS: Our data suggest that CDGP and CHH have distinct genetic profiles, and this finding may facilitate the differential diagnosis in patients presenting with delayed puberty.

Can the diagnosis of NF1 be excluded clinically? A lack of pigmentary findings in families with spinal neurofibromatosis demonstrates a limitation of clinical diagnosis.

BACKGROUND: Consensus clinical diagnostic criteria for neurofibromatosis type I (NF1) include café-au-lait macules and skinfold freckling. The former are frequently the earliest manifestation of NF1, and as such are of particular significance when assessing young children at risk of the condition. A phenotype of predominantly spinal neurofibromatosis has been identified in a small minority of families with NF1, often in association with a relative or absolute lack of cutaneous manifestations. An association with splicing and missense mutations has previously been reported for spinal neurofibromatosis, but on the basis of molecular results in only a few families. METHOD: Patients with spinal NF1 were identified through the Manchester nationally commissioned service for complex NF1. RESULTS: Five families with spinal NF1 were identified, with a broad spectrum of NF1 mutations, providing further evidence that this phenotype may arise in association with any genre of mutation in this gene. Pigmentary manifestations were absent or very mild in affected individuals. Several further affected individuals, some with extensive spinal root tumours, were ascertained when additional family members were assessed. CONCLUSIONS: Clinical NF1 consensus criteria cannot be used to exclude the diagnosis of spinal NF1, especially in childhood. This emphasises the importance of molecular confirmation in individuals and families with atypical presentations of NF1.

Pathogenic mitochondrial tRNA point mutations: nine novel mutations affirm their importance as a cause of mitochondrial disease.

Mutations in the mitochondrial genome, and in particular the mt-tRNAs, are an important cause of human disease. Accurate classification of the pathogenicity of novel variants is vital to allow accurate genetic counseling for patients and their families. The use of weighted criteria based on functional studies-outlined in a validated pathogenicity scoring system--is therefore invaluable in determining whether novel or rare mt-tRNA variants are pathogenic. Here, we describe the identification of nine novel mt--tRNA variants in nine families, in which the probands presented with a diverse range of clinical phenotypes including mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes, isolated progressive external ophthalmoplegia, epilepsy, deafness and diabetes. Each of the variants identified (m.4289T>C, MT-TI; m.5541C>T, MT-TW; m.5690A>G, MT-TN; m.7451A>T, MT-TS1; m.7554G>A, MT-TD; m.8304G>A, MT-TK; m.12206C>T, MT-TH; m.12317T>C, MT-TL2; m.16023G>A, MT-TP) was present in a different tRNA, with evidence in support of pathogenicity, and where possible, details of mutation transmission documented. Through the application of the pathogenicity scoring system, we have classified six of these variants as "definitely pathogenic" mutations (m.5541C>T, m.5690A>G, m.7451A>T, m.12206C>T, m.12317T>C, and m.16023G>A), whereas the remaining three currently lack sufficient evidence and are therefore classed as 'possibly pathogenic' (m.4289T>C, m.7554G>A, and m.8304G>A).

Loss-of-function mutations in SMARCE1 cause an inherited disorder of multiple spinal meningiomas.

One-third of all primary central nervous system tumors in adults are meningiomas. Rarely, meningiomas occur at multiple sites, usually occurring in individuals with type 2 neurofibromatosis (NF2). We sequenced the exomes of three unrelated individuals with familial multiple spinal meningiomas without NF2 mutations. We identified two individuals with heterozygous loss-of-function mutations in the SWI/SNF chromatin-remodeling complex subunit gene SMARCE1. Sequencing of SMARCE1 in six further individuals with spinal meningiomas identified two additional heterozygous loss-of-function mutations. Tumors from individuals with SMARCE1 mutations were of clear-cell histological subtype, and all had loss of SMARCE1 protein, consistent with a tumor suppressor mechanism. Our findings identify multiple-spinal-meningioma disease as a new discrete entity and establish a key role for the SWI/SNF complex in the pathogenesis of both meningiomas and tumors with clear-cell histology.

Craniosynostosis with ectopia lentis and a homozygous 20-base deletion in ADAMTSL4.

Craniosynostosis with ectopia lentis has been described five times since 1950 with unknown inheritance and variable phenotype. The patient was diagnosed with right coronal synostosis at age 10 weeks requiring surgery, and bilateral ectopia lentis with high myopia at 10 months. No other family member was affected. There is no known consanguinity within the family. Genetic screening ruled out FBN1, TGFBR2, and the known craniosynostosis hotspots (FGFR2 exon 8 and exon 10 and FGFR3 exon 6) as the cause. A homozygous deletion in exon 6 of ADAMTSL4 (c.767_786del 20) that has been shown to cause isolated ectopia lentis was found. The mutation results in a premature termination codon (p.Gln256ProfsX38). The proband's mother, father and one sibling are heterozygous carriers of the mutation. This is the first detailed report of a possible genetic determinant of craniosynostosis with ectopia lentis. Although this mutation causes isolated ectopia lentis, this may be evidence of pleiotropic effects of ADAMTSL4 and may represent an overlapping syndrome with a causative mutation in ADAMTSL4. These findings need to be confirmed in further cases with craniosynostosis and ectopia lentis.

A molecular analysis of individuals with neurofibromatosis type 1 (NF1) and optic pathway gliomas (OPGs), and an assessment of genotype-phenotype correlations.

BACKGROUND: Neurofibromatosis type 1 (NF1) affects 1 in 2500 people, and 15% of these may develop an optic pathway glioma (OPG). OPGs behave differently in NF1, and, given their frequency, surveillance is important. However, this is difficult because of the additional complications these patients may have, such as learning difficulties. Management is also different given that NF1 results from loss of function of tumour suppressor gene. A genotype-phenotype correlation may help to determine who is at risk of developing these tumours, aid focused screening, and shed light on response to treatments. METHODS: As part of a long-term follow-up study of patients with NF1 OPGs, the authors assessed genotype-phenotype correlation. Fluorescein in situ hybridisation was performed to identify large deletions, and then a full gene screen for mutations, by denaturing high-performance liquid chromatography. RESULTS: 80 patients with NF1 OPGs were identified, and molecular analyses were performed in a subset of 29. A clustering of pathogenic changes in the 5' tertile of the gene was found. The authors combined these results with those for another two NF1 OPG cohorts and collectively found the same trend. When compared with a control population of NF1 patients without an OPG, the OR of a mutation being present in the 5' tertile was 6.05 (p=0.003) in the NF1 OPG combined cohorts. CONCLUSION: It is possible that genotype is a significant determinant of the risk of development of OPGs in NF1.

Hypophosphatasia: molecular testing of 19 prenatal cases and discussion about genetic counseling.

OBJECTIVE: We studied hypophosphatasia (HP) mutations in 19 cases prenatally detected by ultrasonography without familial history of HP. We correlated the mutations with the reported ultrasound signs, and discussed genetic counseling with regard to the particular dominantly inherited prenatal benign form of HP. METHOD: The coding sequence of the tissue nonspecific alkaline phosphatase (TNSALP) gene was analyzed by DNA sequencing, and 3D modeling was used to locate the mutated amino acids with regard to the functional domains of TNSALP. RESULTS: Although reported ultrasound signs were heterogeneous, two mutated alleles were found in 18 of the 19 cases studied, indicating recessive transmission of the disease. Functional domains of TNSALP were affected by 74% of missense mutations. In all the cases, including one with only a heterozygous mutation, molecular, biological, and familial data do not corroborate the hypothesis of prenatal benign HP. The mutation c.1133A>T observed in the prenatal benign form of HP and common in USA was not found in this series. CONCLUSION: The results point out the prenatally detectable allelic heterogeneity of HP. The nature of the detected mutations and the evidence of recessive inheritance do not support these cases being affected with prenatal benign HP.

Detection of IgE antibodies to bacitracin using a commercially available streptavidin-linked solid phase in a patient with anaphylaxis to triple antibiotic ointment.

BACKGROUND: Bacitracin is a commonly used topical antibiotic that has on occasion been reported to cause anaphylaxis. Evidence of the role of bacitracin specific IgE in such reactions has been demonstrated by skin testing. Because of the potential for provoking a systemic reaction by skin testing, it would be advantageous to develop an in vitro test for bacitracin specific IgE. OBJECTIVE: To report our experience coupling bacitracin to a solid phase and using it to detect specific IgE to bacitracin by fluorescent enzyme immunoassay. METHODS: A patient with a history of recurrent anaphylaxis that developed after application of triple antibiotic ointment to an open wound underwent skin testing with triple antibiotic ointment. Bacitracin was biotinylated and coupled to streptavidin ImmunoCAPs. IgE against bacitracin in the patient's serum was detected by fluorescent enzyme immunoassay. RESULTS: Topical application of triple antibiotic ointment to intact skin produced a 7 X 8-mm wheal with pseudopods. IgE against bacitracin was detected using biotinylated bacitracin-streptavidin ImmunoCAPs at a level of approximately 0.6 KUA/L and confirmed with ImmunoCAPs using direct coupling of bacitracin to the solid phase. CONCLUSIONS: We demonstrate the presence of IgE antibodies to bacitracin in a patient with anaphylaxis to triple antibiotic ointment using a recently described procedure for producing custom allergen solid phases for immunoassay.

Second primary tumors in neurofibromatosis 1 patients treated for optic glioma: substantial risks after radiotherapy.

PURPOSE: Optic pathway gliomas (OPGs) are the most common CNS tumor in neurofibromatosis 1 (NF1) patients. We evaluated the long-term risk of second tumors in NF1-related OPGs after radiotherapy. PATIENTS AND METHODS: We reviewed 80 NF1 OPG patients from two NF1 clinics to evaluate the long-term risk of developing subsequent nervous system tumors, with or without radiotherapy. RESULTS: Fifty-eight patients were assessable for second tumors. Nine (50%) of 18 patients who received radiotherapy after their OPGs developed 12 second tumors in 308 person-years of follow-up after radiotherapy. Eight (20%) of 40 patients who were not treated with radiotherapy developed nine tumors in 721 person-years of follow-up after diagnosis of their OPGs. The relative risk of second nervous system tumor after radiotherapy was 3.04 (95% CI, 1.29 to 7.15). CONCLUSION: There is a significantly increased risk of second nervous system tumors in those NF1 patients who received radiotherapy for their OPGs, especially when treated in childhood. Thus radiotherapy should only be used if absolutely essential in children with NF1.

Characterization of missense mutations and large deletions in the ALPL gene by sequencing and quantitative multiplex PCR of short fragments.

Hypophosphatasia is a rare inherited bone disorder characterized by defective bone and dental mineralization and deficiency of serum and liver/bone/kidney alkaline phosphatase activity. The disease is due to mutations in the alkaline phosphatase liver-type (ALPL) gene. Gross deletions or insertions have not previously been reported in this gene. We report here the characterization of nine novel ALPL gene mutations in a series of 8 patients affected by various forms of hypophosphatasia. The newly discovered mutations included five missense mutations (c.368C --> A, c.814C--> T, c.1196C--> T, c.1199C--> T, c.1283G--> C), two small deletions (c.797_802del, c.1044_1055del), and two large deletions. The large deletions were detected by quantitative multiplex polymerase chain reaction (PCR) of short fluorescent fragments (QMPSF). We conclude that QMPSF slightly reduces the proportion of undetected mutations in hypophosphatasia and improves genetic counselling in the affected families.