Short-read whole genome sequencing identifies causative variants in most individuals with previously unexplained aniridia.

BACKGROUND: Classic aniridia is a highly penetrant autosomal dominant disorder characterised by congenital absence of the iris, foveal hypoplasia, optic disc anomalies and progressive opacification of the cornea. >90% of cases of classic aniridia are caused by heterozygous, loss-of-function variants affecting the PAX6 locus. METHODS: Short-read whole genome sequencing was performed on 51 (39 affected) individuals from 37 different families who had screened negative for mutations in the PAX6 coding region. RESULTS: Likely causative mutations were identified in 22 out of 37 (59%) families. In 19 out of 22 families, the causative genomic changes have an interpretable deleterious impact on the PAX6 locus. Of these 19 families, 1 has a novel heterozygous PAX6 frameshift variant missed on previous screens, 4 have single nucleotide variants (SNVs) (one novel) affecting essential splice sites of PAX6 5' non-coding exons and 2 have deep intronic SNV (one novel) resulting in gain of a donor splice site. In 12 out of 19, the causative variants are large-scale structural variants; 5 have partial or whole gene deletions of PAX6, 3 have deletions encompassing critical PAX6 cis-regulatory elements, 2 have balanced inversions with disruptive breakpoints within the PAX6 locus and 2 have complex rearrangements disrupting PAX6. The remaining 3 of 22 families have deletions encompassing FOXC1 (a known cause of atypical aniridia). Seven of the causative variants occurred de novo and one cosegregated with familial aniridia. We were unable to establish inheritance status in the remaining probands. No plausibly causative SNVs were identified in PAX6 cis-regulatory elements. CONCLUSION: Whole genome sequencing proves to be an effective diagnostic test in most individuals with previously unexplained aniridia.

Heterozygous variants in ZBTB7A cause a neurodevelopmental disorder associated with symptomatic overgrowth of pharyngeal lymphoid tissue, macrocephaly, and elevated fetal hemoglobin.

By clinical whole exome sequencing, we identified 12 individuals with ages 3 to 37 years, including three individuals from the same family, with a consistent phenotype of intellectual disability (ID), macrocephaly, and overgrowth of adenoid tissue. All 12 individuals harbored a rare heterozygous variant in ZBTB7A which encodes the transcription factor Zinc finger and BTB-domain containing protein 7A, known to play a role in lympho- and hematopoiesis. ID was generally mild. Fetal hemoglobin (HbF) fraction was elevated 2.2%-11.2% (reference value <2% in individuals > 6 months) in four of the five individuals for whom results were available. Ten of twelve individuals had undergone surgery at least once for lymphoid hypertrophy limited to the pharynx. In the most severely affected individual (individual 1), airway obstruction resulted in 17 surgical procedures before the age of 13 years. Sleep apnea was present in 8 of 10 individuals. In the nine unrelated individuals, ZBTB7A variants were novel and de novo. The six frameshift/nonsense and four missense variants were spread throughout the gene. This is the first report of a cohort of individuals with this novel syndromic neurodevelopmental disorder.

De Novo Mutations in Protein Kinase Genes CAMK2A and CAMK2B Cause Intellectual Disability.

Calcium/calmodulin-dependent protein kinase II (CAMK2) is one of the first proteins shown to be essential for normal learning and synaptic plasticity in mice, but its requirement for human brain development has not yet been established. Through a multi-center collaborative study based on a whole-exome sequencing approach, we identified 19 exceedingly rare de novo CAMK2A or CAMK2B variants in 24 unrelated individuals with intellectual disability. Variants were assessed for their effect on CAMK2 function and on neuronal migration. For both CAMK2A and CAMK2B, we identified mutations that decreased or increased CAMK2 auto-phosphorylation at Thr286/Thr287. We further found that all mutations affecting auto-phosphorylation also affected neuronal migration, highlighting the importance of tightly regulated CAMK2 auto-phosphorylation in neuronal function and neurodevelopment. Our data establish the importance of CAMK2A and CAMK2B and their auto-phosphorylation in human brain function and expand the phenotypic spectrum of the disorders caused by variants in key players of the glutamatergic signaling pathway.

Primary immunodeficiency diseases: Genomic approaches delineate heterogeneous Mendelian disorders.

BACKGROUND: Primary immunodeficiency diseases (PIDDs) are clinically and genetically heterogeneous disorders thus far associated with mutations in more than 300 genes. The clinical phenotypes derived from distinct genotypes can overlap. Genetic etiology can be a prognostic indicator of disease severity and can influence treatment decisions. OBJECTIVE: We sought to investigate the ability of whole-exome screening methods to detect disease-causing variants in patients with PIDDs. METHODS: Patients with PIDDs from 278 families from 22 countries were investigated by using whole-exome sequencing. Computational copy number variant (CNV) prediction pipelines and an exome-tiling chromosomal microarray were also applied to identify intragenic CNVs. Analytic approaches initially focused on 475 known or candidate PIDD genes but were nonexclusive and further tailored based on clinical data, family history, and immunophenotyping. RESULTS: A likely molecular diagnosis was achieved in 110 (40%) unrelated probands. Clinical diagnosis was revised in about half (60/110) and management was directly altered in nearly a quarter (26/110) of families based on molecular findings. Twelve PIDD-causing CNVs were detected, including 7 smaller than 30 Kb that would not have been detected with conventional diagnostic CNV arrays. CONCLUSION: This high-throughput genomic approach enabled detection of disease-related variants in unexpected genes; permitted detection of low-grade constitutional, somatic, and revertant mosaicism; and provided evidence of a mutational burden in mixed PIDD immunophenotypes.

Two male sibs with severe micrognathia and a missense variant in MED12.

Missense variants in MED12 cause three partially overlapping dysmorphic X-linked intellectual disability (XLID) syndromes: Lujan-Fryns syndrome (also known as Lujan syndrome), FG syndrome (also known as Opitz-Kaveggia syndrome) and X-linked Ohdo syndrome. We report a family with two severely micrognathic male sibs, a 10½ year old boy and a fetus, in which hemizygosity for a previously unreported missense variant in exon 13 of MED12 (NM_005120.2), c.1862G > A, p.(Arg621Gln) was detected by whole exome sequencing. The affected sibs shared no other rare variant with relevance to the phenotype. X-chromosome inactivation in blood was completely skewed (100:0) in the unaffected heterozygous mother, most likely as a result of preferential inactivation of the X-chromosome harbouring the missense variant in MED12. Neither the unaffected brother nor the unaffected maternal grandfather carried the missense variant in MED12. In the 10½ year old boy, upper airway obstruction secondary to Pierre Robin sequence necessitated a tracheostomy for the first 10 months of life. He has mild to moderate intellectual disability and some dysmorphic features seen in MED12-related syndromes. In addition, he has a horizontal gaze paresis, anomalies of the inner ear, and a cervical block vertebra. This report contributes to the expanding phenotypic range associated with MED12-mutations.

Genetic Analysis of 'PAX6-Negative' Individuals with Aniridia or Gillespie Syndrome.

We report molecular genetic analysis of 42 affected individuals referred with a diagnosis of aniridia who previously screened as negative for intragenic PAX6 mutations. Of these 42, the diagnoses were 31 individuals with aniridia and 11 individuals referred with a diagnosis of Gillespie syndrome (iris hypoplasia, ataxia and mild to moderate developmental delay). Array-based comparative genomic hybridization identified six whole gene deletions: four encompassing PAX6 and two encompassing FOXC1. Six deletions with plausible cis-regulatory effects were identified: five that were 3' (telomeric) to PAX6 and one within a gene desert 5' (telomeric) to PITX2. Sequence analysis of the FOXC1 and PITX2 coding regions identified two plausibly pathogenic de novo FOXC1 missense mutations (p.Pro79Thr and p.Leu101Pro). No intragenic mutations were detected in PITX2. FISH mapping in an individual with Gillespie-like syndrome with an apparently balanced X;11 reciprocal translocation revealed disruption of a gene at each breakpoint: ARHGAP6 on the X chromosome and PHF21A on chromosome 11. In the other individuals with Gillespie syndrome no mutations were identified in either of these genes, or in HCCS which lies close to the Xp breakpoint. Disruption of PHF21A has previously been implicated in the causation of intellectual disability (but not aniridia). Plausibly causative mutations were identified in 15 out of 42 individuals (12/32 aniridia; 3/11 Gillespie syndrome). Fourteen of these mutations presented in the known aniridia genes; PAX6, FOXC1 and PITX2. The large number of individuals in the cohort with no mutation identified suggests greater locus heterogeneity may exist in both isolated and syndromic aniridia than was previously appreciated.

Biallelic Mutations in UNC80 Cause Persistent Hypotonia, Encephalopathy, Growth Retardation, and Severe Intellectual Disability.

Ion channel proteins are required for both the establishment of resting membrane potentials and the generation of action potentials. Hundreds of mutations in genes encoding voltage-gated ion channels responsible for action potential generation have been found to cause severe neurological diseases. In contrast, the roles of voltage-independent "leak" channels, important for the establishment and maintenance of resting membrane potentials upon which action potentials are generated, are not well established in human disease. UNC80 is a large component of the NALCN sodium-leak channel complex that regulates the basal excitability of the nervous system. Loss-of-function mutations of NALCN cause infantile hypotonia with psychomotor retardation and characteristic facies (IHPRF). We report four individuals from three unrelated families who have homozygous missense or compound heterozygous truncating mutations in UNC80 and persistent hypotonia, encephalopathy, growth failure, and severe intellectual disability. Compared to control cells, HEK293T cells transfected with an expression plasmid containing the c.5098C>T (p.Pro1700Ser) UNC80 mutation found in one individual showed markedly decreased NALCN channel currents. Our findings demonstrate the fundamental significance of UNC80 and basal ionic conductance to human health.

A 5.8 kb deletion removing the entire MNX1 gene in a Norwegian family with Currarino syndrome.

Currarino syndrome (CS) is a clinically variable disorder characterized by anorectal, sacral and presacral anomalies. It is associated with loss-of-function mutations in the motor neuron and pancreas homeobox 1 (MNX1) gene. Inheritance is autosomal dominant, expression variable and penetrance incomplete. We describe a Norwegian family with typical CS in which a heterozygous deletion removes the entire MNX1 gene but no other known genes. We also report MNX1 mutations in three other Norwegian families and confirm that the GCC12 repeat (c.373_375[12]) is a normal allelic variant. This work underscores the importance of dosage analysis of MNX1 when Sanger sequencing is negative.

Mutations in CCBE1 cause generalized lymph vessel dysplasia in humans.

Lymphedema, lymphangiectasias, mental retardation and unusual facial characteristics define the autosomal recessive Hennekam syndrome. Homozygosity mapping identified a critical chromosomal region containing CCBE1, the human ortholog of a gene essential for lymphangiogenesis in zebrafish. Homozygous and compound heterozygous mutations in seven subjects paired with functional analysis in a zebrafish model identify CCBE1 as one of few genes causing primary generalized lymph-vessel dysplasia in humans.

[Molecular genetic diagnostics in syndromes associated with the RAS/MAPK signalling pathway]. / Mutasjonsdiagnostikk ved syndromer knyttet til RAS/MAPK-signalveien.

BACKGROUND: Mutations in genes of the mitogen-activated protein kinase (MAPK) cascade have recently been shown to cause several syndromes characterized by dysmorphic facial features, growth retardation, cognitive impairment, heart disease and cutaneous abnormalities. This signalling pathway involves RAS and RAF proteins, and is central in the regulation of normal growth and the development of cancer. MATERIAL AND METHODS: We have studied 23 Norwegian patients for whom there was a clinical suspicion of Costello, Noonan or cardio-facio-cutaneous syndrome. Patients suspected of having Noonan syndrome had previously tested negative for mutations in the tyrosine phosphatase gene PTPN11. The material was examined for mutations in the HRAS, KRAS, RAF1 and BRAF genes. Two patients are described to illustrate diagnostic challenges and the usefulness of genetic testing. RESULTS: Ten of 23 patients (43 %) had mutations affecting the RAS/MAPK signalling pathway. Mutations in HRAS were most common (five cases), while three patients had mutations in KRAS and two in RAF1. Spontaneous mutations were demonstrated in eight cases. Our data indicate an annual incidence of 1-2 new cases of congenital RAS/RAF mutations in Norway. INTERPRETATION: Upon clinical suspicion of syndromes of the RAS/MAPK signalling pathway, molecular genetic analyses may be essential for a correct diagnosis. Certain mutations are associated with an increased cancer risk, exemplifying that results of genetic laboratory testing may influence medical management.

Two brothers with a microduplication including the MECP2 gene: rapid head growth in infancy and resolution of susceptibility to infection.

Microduplications in chromosome Xq28, which include the methyl-CPG binding protein (MECP2) gene, cause severe X-linked mental retardation. Serious recurrent infections are a feature of this condition. Affected males are micro or normocephalic. We report two normocephalic brothers with an approximately 0.5 Mb duplication which includes MECP2 who had rapid head growth in infancy. The younger boy had chronic constipation until the age of 3 years. For both boys, the susceptibility to infection subsided in the second year of life. Whether or not rapid head growth in infancy and/or constipation are frequent features of the phenotype remains to be seen as more patients are described. Susceptibility to infection can remit after early childhood and could theoretically be related to overexpression of the interleukin 1 receptor-associated kinase IRAK1 gene.

Investigations as a prerequisite for genetic counseling after termination of pregnancy based on sonographic detection of serious central nervous system--or skeletal anomalies.

OBJECTIVES: The primary aim was to evaluate which investigation performed after sonographic detection of central nervous system (CNS) or skeletal anomalies that had highest diagnostic yield. The secondary aim was to estimate recurrence risk. Design. Retrospective review of patients' records. SETTING: Tertiary fetal medicine referral center. SAMPLE: Pregnancy terminations (n=97) because of CNS or skeletal anomalies during a 17-year period, within 12-24 weeks gestation. METHODS: Two medical geneticists and one genetic counselor reviewed charts independently. MAIN OUTCOME MEASURES: Primary ultrasound diagnosis, change in diagnosis following supplementary examinations in addition to prenatal ultrasound (medical history, autopsy, post-mortem X-ray, karyotyping, targeted DNA analysis and investigations for infection), the most useful method to determine diagnosis, and recurrence risk estimate including inter-rater agreement. RESULTS: Mean gestational age was 19.8 weeks. All three investigators agreed in each case on which investigation constituted the best basis to determine the most precise diagnosis. The examinations performed in addition to prenatal ultrasound provided important diagnostic information in 54 cases (56%) and altered recurrence risk in 22 (23%) cases; in eight of these cases the risk estimate was increased. In nine cases (9%) the investigators disagreed in their estimates of recurrence risk. Kappa for inter-rater agreement was >0.90. CONCLUSIONS: A panel of diagnostic investigations, depending on the organ system involved, allows for a more precise diagnosis and a more reliable estimate of recurrence risk than prenatal ultrasound alone. In some instances, recurrence risk estimation is not straightforward as evidenced by lack of consensus.

Distal phalangeal creases--a distinctive dysmorphic feature in disorders of the RAS signalling pathway?

We have observed unusual transverse distal phalangeal creases in two patients, one with Costello syndrome (G12S mutation in the HRAS gene) and one with cardio-facio-cutaneous (CFC) syndrome or possibly Noonan syndrome (Q22E mutation in the KRAS gene). This feature along with fetal pads was present in both children at birth and has persisted until age two years. Distal phalangeal creases, when present, may be a good diagnostic handle for syndromes belonging to the RAS signalling pathway.

Posterior helical pits.

Posterior helical pits are an easily overlooked finding. They are uncommon in the general population although they do occur as an isolated autosomal dominant trait. We have observed posterior helical pits as a feature in three syndromes only: Beckwith-Wiedemann syndrome, Simpson-Golabi-Behmel syndrome and Rubinstein-Taybi syndrome. We suggest that posterior helical pits, when present, are an excellent diagnostic handle.

A surrealistic mega-analysis of redisorganization theories.

BACKGROUND: We are sick and tired of being redisorganized. OBJECTIVE: To systematically review the empirical evidence for organizational theories and repeated reorganizations. METHODS: We did not find anything worth reading, other than Dilbert, so we fantasized. Unfortunately, our fantasies may well resemble many people's realities. We are sorry about this, but it is not our fault. RESULTS: We discovered many reasons for repeated reorganizations, the most common being 'no good reason'. We estimated that trillions of dollars are being spent on strategic and organizational planning activities each year, thus providing lots of good reasons for hundreds of thousands of people, including us, to get into the business. New leaders who are intoxicated with the prospect of change further fuel perpetual cycles of redisorganization. We identified eight indicators of successful redisorganizations, including large consultancy fees paid to friends and relatives. CONCLUSIONS: We propose the establishment of ethics committees to review all future redisorganization proposals in order to put a stop to uncontrolled, unplanned experimentation inflicted providers and users of the health services.