Hemizygous variants in protein phosphatase 1 regulatory subunit 3F (PPP1R3F) are associated with a neurodevelopmental disorder characterized by developmental delay, intellectual disability and autistic features.

Protein phosphatase 1 regulatory subunit 3F (PPP1R3F) is a member of the glycogen targeting subunits (GTSs), which belong to the large group of regulatory subunits of protein phosphatase 1 (PP1), a major eukaryotic serine/threonine protein phosphatase that regulates diverse cellular processes. Here, we describe the identification of hemizygous variants in PPP1R3F associated with a novel X-linked recessive neurodevelopmental disorder in 13 unrelated individuals. This disorder is characterized by developmental delay, mild intellectual disability, neurobehavioral issues such as autism spectrum disorder, seizures and other neurological findings including tone, gait and cerebellar abnormalities. PPP1R3F variants segregated with disease in affected hemizygous males that inherited the variants from their heterozygous carrier mothers. We show that PPP1R3F is predominantly expressed in brain astrocytes and localizes to the endoplasmic reticulum in cells. Glycogen content in PPP1R3F knockout astrocytoma cells appears to be more sensitive to fluxes in extracellular glucose levels than in wild-type cells, suggesting that PPP1R3F functions in maintaining steady brain glycogen levels under changing glucose conditions. We performed functional studies on nine of the identified variants and observed defects in PP1 binding, protein stability, subcellular localization and regulation of glycogen metabolism in most of them. Collectively, the genetic and molecular data indicate that deleterious variants in PPP1R3F are associated with a new X-linked disorder of glycogen metabolism, highlighting the critical role of GTSs in neurological development. This research expands our understanding of neurodevelopmental disorders and the role of PP1 in brain development and proper function.

Familial acute aortic dissection associated with a novel ACTA2 germline variant.

Aortic dissection is a life-threatening cardiovascular disease. Hereditary disorders are responsible for a small percentage of cases. Nonetheless, it is important to identify genetic causes, as they are often autosomal dominantly inherited and are of life-saving importance if we can identify persons at risk. Mutations of the ACTA2 gene are the most common cause of non-syndromic familial aortic disease. Exploration of the genetic background in suspected familial cases and determination of the exact etiology are mandatory for management and establishing appropriate follow-up strategies due to the risk of fatal recurrences. Herein, we present a 21-year-old male with a familial acute aortic dissection associated with novel ACTA2 germline variant and discuss the management and surveillance considerations.

Low-level mosaicism in tuberous sclerosis complex in four unrelated patients: Comparison of clinical characteristics and diagnostic pathways.

Tuberous sclerosis complex (TSC) is an autosomal dominant neurocutaneous syndrome caused by either TSC1 or TSC2 gene mutations. About 15% of TSC patients remain without genetic diagnosis by conventional analysis despite clinical evidence. It is important to identify somatic mosaics, as therapeutic options are now available in patients with TSC1 or TSC2 mutations. Here, we describe the clinical and genetic characteristics of four male TSC patients with low-level mosaicism. Patients presented at ages between 9 months and 32 years. Clinical manifestations varied considerably and included brain lesions in all four patients, cardiac rhabdomyomas in two young patients, skin involvement in two patients, and retinal hamartomas and renal angiomyolipomas in three patients. One patient presented with epileptic seizures and psychomotor delay. Low levels of mosaicism for TSC1 or TSC2 mutation were found in different tissue samples employing next generation sequencing and multiple ligation-dependent probe amplification. The five disease-associated variants, including one second-hit mutation, include three truncating mutations and one deletion in TSC2, and one truncating mutation in TSC1. Sanger sequencing, allele-specific oligonucleotide PCR (ASO-PCR), and droplet digital PCR were used to confirm and quantify the disclosed mutations. Genetic identification of low-level mosaicism for TSC remains challenging but is important for optimal surveillance and management.

Mutations in HID1 Cause Syndromic Infantile Encephalopathy and Hypopituitarism.

OBJECTIVE: Precursors of peptide hormones undergo posttranslational modifications within the trans-Golgi network (TGN). Dysfunction of proteins involved at different steps of this process cause several complex syndromes affecting the central nervous system (CNS). We aimed to clarify the genetic cause in a group of patients characterized by hypopituitarism in combination with brain atrophy, thin corpus callosum, severe developmental delay, visual impairment, and epilepsy. METHODS: Whole exome sequencing was performed in seven individuals of six unrelated families with these features. Postmortem histopathological and HID1 expression analysis of brain tissue and pituitary gland were conducted in one patient. Functional consequences of the homozygous HID1 variant p.R433W were investigated by Seahorse XF Assay in fibroblasts of two patients. RESULTS: Bi-allelic variants in the gene HID1 domain-containing protein 1 (HID1) were identified in all patients. Postmortem examination confirmed cerebral atrophy with enlarged lateral ventricles. Markedly reduced expression of pituitary hormones was found in pituitary gland tissue. Colocalization of HID1 protein with the TGN was not altered in fibroblasts of patients compared to controls, while the extracellular acidification rate upon stimulation with potassium chloride was significantly reduced in patient fibroblasts compared to controls. INTERPRETATION: Our findings indicate that mutations in HID1 cause an early infantile encephalopathy with hypopituitarism as the leading presentation, and expand the list of syndromic CNS diseases caused by interference of TGN function. ANN NEUROL 2021;90:149-164.

The genetic landscape of axonal neuropathies in the middle-aged and elderly: Focus on MME.

OBJECTIVE: To test the hypothesis that monogenic neuropathies such as Charcot-Marie-Tooth disease (CMT) contribute to frequent but often unexplained neuropathies in the elderly, we performed genetic analysis of 230 patients with unexplained axonal neuropathies and disease onset ≥35 years. METHODS: We recruited patients, collected clinical data, and conducted whole-exome sequencing (WES; n = 126) and MME single-gene sequencing (n = 104). We further queried WES repositories for MME variants and measured blood levels of the MME-encoded protein neprilysin. RESULTS: In the WES cohort, the overall detection rate for assumed disease-causing variants in genes for CMT or other conditions associated with neuropathies was 18.3% (familial cases 26.4%, apparently sporadic cases 12.3%). MME was most frequently involved and accounted for 34.8% of genetically solved cases. The relevance of MME for late-onset neuropathies was further supported by detection of a comparable proportion of cases in an independent patient sample, preponderance of MME variants among patients compared to population frequencies, retrieval of additional late-onset neuropathy patients with MME variants from WES repositories, and low neprilysin levels in patients' blood samples. Transmission of MME variants was often consistent with an incompletely penetrant autosomal-dominant trait and less frequently with autosomal-recessive inheritance. CONCLUSIONS: A detectable fraction of unexplained late-onset axonal neuropathies is genetically determined, by variants in either CMT genes or genes involved in other conditions that affect the peripheral nerves and can mimic a CMT phenotype. MME variants can act as completely penetrant recessive alleles but also confer dominantly inherited susceptibility to axonal neuropathies in an aging population.

A Syrian patient with Steel syndrome due to compound heterozygous COL27A1 mutations with colobomata of the eye.

The joint occurrence of short stature, congenital dislocation of the hip, carpal coalition, dislocation of the radial head, cavus deformity, scoliosis, and vertebral anomalies was first described in 1993 by Steel et al. (OMIM #615155) in 23 children from Puerto Rico. The condition is caused by a deficient matrix protein, collagen type XXVII alpha 1 chain, due to bi-allelic loss of function mutations in the gene COL27A1. Outside of Puerto Rico, only four families have been described, in three of which the patients also had hearing loss. However, structural eye defects have not yet been reported in conjunction with this rare autosomal recessive syndrome. Here, we describe a 9-year-old girl born to nonconsanguineous Syrian parents with the characteristic features of Steel syndrome, including short stature, massive malalignment of large joints, kyphoscoliosis, hearing loss, and typical facial dysmorphism. However, she was also born with bilateral colobomata of the irides and choroido-retinae with unilateral affection of the macula. Whole exome sequencing identified two pathogenic compound heterozygous variants in COL27A1: c.93del, p.(Phe32Leufs*71) and c.3075del, p.(Lys1026Argfs*33). There was no discernible alternative cause for the colobomata. Our findings might indicate an association of this exceptionally rare disorder caused by COL27A1 mutations with developmental defects of the eye from the anophthalmia/microphthalmia/coloboma spectrum.

Diagnosis and Therapy Before Assisted Reproductive Treatments. Guideline of the DGGG, OEGGG and SGGG (S2k Level, AWMF Register Number 015-085, February 2019) - Part 1, Basic Assessment of the Woman.

Introduction Supporting and counselling couples with fertility issues prior to starting ART is a multidisciplinary diagnostic and therapeutic challenge. The first German/Austrian/Swiss interdisciplinary S2k guideline on "Diagnosis and Therapy Before Assisted Reproductive Treatments (ART)" was published in February 2019. This guideline was developed in the context of the guidelines program of the German Society of Gynecology and Obstetrics (DGGG) in cooperation with the Swiss Society of Gynecology and Obstetrics (SGGG) and the Austrian Society of Gynecology and Obstetrics (OEGGG). Aims One third of the causes of involuntary childlessness are still unclear, even if the woman or man have numerous possible risk factors. Because the topic is still very much taboo, couples may be socially isolated and often only present quite late to a fertility center. At present, there is no standard treatment concept, as currently no standard multidisciplinary procedures exist for the diagnostic workup and treatment of infertility. The aim of this guideline is to provide physicians with evidence-based recommendations for counselling, diagnostic workup and treatment. Methods This S2k guideline was developed on behalf of the Guidelines Commission of the DGGG by representative members from different professional medical organizations and societies using a structured consensus process. Recommendations The first part of this guideline focuses on the basic assessment of affected women, including standard anatomical and endocrinological diagnostic procedures and examinations into any potential infections. Other areas addressed in this guideline are the immunological workup with an evaluation of the patient's vaccination status, an evaluation of psychological factors, and the collection of data relating to other relevant factors affecting infertility. The second part will focus on explanations of diagnostic procedures compiled in collaboration with specialists from other medical specialties such as andrologists, human geneticists and oncologists.

Diagnosis and Treatment Before Assisted Reproductive Treatments. Guideline of the DGGG, OEGGG and SGGG (S2k Level, AWMF Register Number 015-085, February 2019) - Part 2, Hemostaseology, Andrology, Genetics and History of Malignant Disease.

Introduction Supporting and counselling couples with fertility issues prior to starting ART is a multidisciplinary diagnostic and therapeutic challenge. The first German-language interdisciplinary S2k guideline on "Diagnosis and Therapy Before Assisted Reproductive Treatments (ART)" was published in February 2019. The guideline was developed in the context of the guidelines program of the German Society of Gynecology and Obstetrics (DGGG) in cooperation with the Swiss Society of Gynecology and Obstetrics (SGGG) and the Austrian Society of Gynecology and Obstetrics (OEGGG). Aim In one third of cases, the cause of involuntary childlessness remains unclear, even if the woman or man have numerous possible risk factors. Because the topic is still very much taboo, couples may be socially isolated and often only present quite late to a fertility center. There is no standard treatment concept for these patients at present, as there are currently no standard multidisciplinary procedures for the diagnostic workup and treatment of infertility. The aim of this guideline is to provide physicians with evidence-based recommendations for counselling, diagnosis and treatment. Methods This S2k guideline was developed on behalf of the Guidelines Commission of the DGGG by representative members from different professional medical organizations and societies using a structured consensus process. Recommendations This second part of the guideline describes the hematological workup for women as well as additional diagnostic procedures which can be used to investigate couples and which are carried out in cooperation with physicians working in other medical fields such as andrologists, geneticists and oncologists.

Early onset facioscapulohumeral muscular dystrophy - Long-term follow-up of a patient with total facial diplegia.

We report a patient with early onset facioscapulohumeral muscular dystrophy type 1 (FSHD1) who was not diagnosed until 48 years of age. She developed progressive facial diplegia from the age of 4-5 years followed by limb muscle weakness. Motor nerve conduction was normal, myopathic changes were seen electromyographically. Creatine kinase activity was mildly increased at the beginning. Muscle biopsy at 8 years suggested a neurogenic pattern, a second biopsy at age 30 was chronic myopathic with fibre calibre variation. The patient lost the ability to walk at age 44. When last seen she had total facial diplegia, no active movements in her limbs, mild kyphoscoliosis and a rigid thoracic spine. Molecular studies revealed a shortened D4Z4 fragment confirming the diagnosis FSHD1. Her family history was unremarkable, suggesting a de novo mutation. This report is to illustrate the evolving phenotype of early onset FSHD1 with predominating facial palsy.

Loss of tubulin deglutamylase CCP1 causes infantile-onset neurodegeneration.

A set of glutamylases and deglutamylases controls levels of tubulin polyglutamylation, a prominent post-translational modification of neuronal microtubules. Defective tubulin polyglutamylation was first linked to neurodegeneration in the Purkinje cell degeneration (pcd) mouse, which lacks deglutamylase CCP1, displays massive cerebellar atrophy, and accumulates abnormally glutamylated tubulin in degenerating neurons. We found biallelic rare and damaging variants in the gene encoding CCP1 in 13 individuals with infantile-onset neurodegeneration and confirmed the absence of functional CCP1 along with dysregulated tubulin polyglutamylation. The human disease mainly affected the cerebellum, spinal motor neurons, and peripheral nerves. We also demonstrate previously unrecognized peripheral nerve and spinal motor neuron degeneration in pcd mice, which thus recapitulated key features of the human disease. Our findings link human neurodegeneration to tubulin polyglutamylation, entailing this post-translational modification as a potential target for drug development for neurodegenerative disorders.

Evidence of mild founder LMOD3 mutations causing nemaline myopathy 10 in Germany and Austria.

OBJECTIVE: To expand the clinical and genetic spectrum of nemaline myopathy 10 by a series of Austrian and German patients with a milder disease course and missense mutations in LMOD3. METHODS: We characterized the clinical features and the genetic status of 4 unrelated adolescent or adult patients with nemaline myopathy. RESULTS: The 4 patients showed a relatively mild disease course. They all have survived into adulthood, 3 of 4 have remained ambulatory, and all showed marked facial weakness. Muscle biopsy specimens gave evidence of nemaline bodies. All patients were unrelated but originated from Austria (Tyrol and Upper Austria) and Southern Germany (Bavaria). All patients carried the missense variant c.1648C>T, p.(Leu550Phe) in the LMOD3 gene, either on both alleles or in trans with another missense variant (c.1004A>G, p.Gln335Arg). Both variants were not reported previously. CONCLUSIONS: In 2014, a severe form of congenital nemaline myopathy caused by disrupting mutations in LMOD3 was identified and denoted as NEM10. Unlike the previously reported patients, who had a severe clinical picture with a substantial risk of early death, our patients showed a relatively mild disease course. As the missense variant c.1648C>T is located further downstream compared to all previously published LMOD3 mutations, it might be associated with higher protein expression compared to the reported loss-of-function mutations. The apparent clusters of 2 mild mutations in Germany and Austria in 4 unrelated families may be explained by a founder effect.

Rare Variants in MME, Encoding Metalloprotease Neprilysin, Are Linked to Late-Onset Autosomal-Dominant Axonal Polyneuropathies.

Axonal polyneuropathies are a frequent cause of progressive disability in the elderly. Common etiologies comprise diabetes mellitus, paraproteinaemia, and inflammatory disorders, but often the underlying causes remain elusive. Late-onset axonal Charcot-Marie-Tooth neuropathy (CMT2) is an autosomal-dominantly inherited condition that manifests in the second half of life and is genetically largely unexplained. We assumed age-dependent penetrance of mutations in a so far unknown gene causing late-onset CMT2. We screened 51 index case subjects with late-onset CMT2 for mutations by whole-exome (WES) and Sanger sequencing and subsequently queried WES repositories for further case subjects carrying mutations in the identified candidate gene. We studied nerve pathology and tissue levels and function of the abnormal protein in order to explore consequences of the mutations. Altogether, we observed heterozygous rare loss-of-function and missense mutations in MME encoding the metalloprotease neprilysin in 19 index case subjects diagnosed with axonal polyneuropathies or neurodegenerative conditions involving the peripheral nervous system. MME mutations segregated in an autosomal-dominant fashion with age-related incomplete penetrance and some affected individuals were isolated case subjects. We also found that MME mutations resulted in strongly decreased tissue availability of neprilysin and impaired enzymatic activity. Although neprilysin is known to degrade ß-amyloid, we observed no increased amyloid deposition or increased incidence of dementia in individuals with MME mutations. Detection of MME mutations is expected to increase the diagnostic yield in late-onset polyneuropathies, and it will be tempting to explore whether substances that can elevate neprilysin activity could be a rational option for treatment.

Mutations in Subunits of the Activating Signal Cointegrator 1 Complex Are Associated with Prenatal Spinal Muscular Atrophy and Congenital Bone Fractures.

Transcriptional signal cointegrators associate with transcription factors or nuclear receptors and coregulate tissue-specific gene transcription. We report on recessive loss-of-function mutations in two genes (TRIP4 and ASCC1) that encode subunits of the nuclear activating signal cointegrator 1 (ASC-1) complex. We used autozygosity mapping and whole-exome sequencing to search for pathogenic mutations in four families. Affected individuals presented with prenatal-onset spinal muscular atrophy (SMA), multiple congenital contractures (arthrogryposis multiplex congenita), respiratory distress, and congenital bone fractures. We identified homozygous and compound-heterozygous nonsense and frameshift TRIP4 and ASCC1 mutations that led to a truncation or the entire absence of the respective proteins and cosegregated with the disease phenotype. Trip4 and Ascc1 have identical expression patterns in 17.5-day-old mouse embryos with high expression levels in the spinal cord, brain, paraspinal ganglia, thyroid, and submandibular glands. Antisense morpholino-mediated knockdown of either trip4 or ascc1 in zebrafish disrupted the highly patterned and coordinated process of α-motoneuron outgrowth and formation of myotomes and neuromuscular junctions and led to a swimming defect in the larvae. Immunoprecipitation of the ASC-1 complex consistently copurified cysteine and glycine rich protein 1 (CSRP1), a transcriptional cofactor, which is known to be involved in spinal cord regeneration upon injury in adult zebrafish. ASCC1 mutant fibroblasts downregulated genes associated with neurogenesis, neuronal migration, and pathfinding (SERPINF1, DAB1, SEMA3D, SEMA3A), as well as with bone development (TNFRSF11B, RASSF2, STC1). Our findings indicate that the dysfunction of a transcriptional coactivator complex can result in a clinical syndrome affecting the neuromuscular system.

Clinical and morphological variability of the E396K mutation in the neurofilament light chain gene in patients with Charcot-Marie- Tooth disease type 2E.

Mutations in the neurofilament light chain (NEFL) gene mostly cause autosomal dominant axonal Charcot-Marie- Tooth neuropathy (CMT2E). The mutation c.1186G>A, p.E396K has been reported in seven unrelated families so far, however, the phenotypic spectrum has not been fully elucidated. Here we describe nine patients with the E396K mutation who had a strikingly discordant clinical severity. The clinical picture in family I (patients I,1-II,8) was characterized by childhood onset, distal and proximal pareses, and loss of ambulation in the 6th decade of life, whereas onset was at age 50 years in patient 9, who had no affected relatives. Electrophysiology and sural nerve biopsy revealed a mixed axonal and demyelinating neuropathy, along with probably coincidental inflammatory small vessel disease in patient 9. Biopsy results in family I suggest that not only axons but also Schwann cells may be primary disease targets in CMT2E. Considerably elevated CK levels in all affected adults of family I as well as pronounced myopathic changes in skeletal muscle biopsies point towards an accompanying muscle involvement as a primary target. Our findings reveal an extended phenotype of CMT2E caused by an identical missense mutation of the NEFL gene.

Congenital mirror movements: mutational analysis of RAD51 and DCC in 26 cases.

OBJECTIVE: We screened a large series of individuals with congenital mirror movements (CMM) for mutations in the 2 identified causative genes, DCC and RAD51. METHODS: We studied 6 familial and 20 simplex CMM cases. Each patient had a standardized neurologic assessment. Analysis of DCC and RAD51 coding regions included Sanger sequencing and a quantitative method allowing detection of micro rearrangements. We then compared the frequency of rare variants predicted to be pathogenic by either the PolyPhen-2 or the SIFT algorithm in our population and in the 4,300 controls of European origin on the Exome Variant Server. RESULTS: We found 3 novel truncating mutations of DCC that segregate with CMM in 4 of the 6 families. Among the 20 simplex cases, we found one exonic deletion of DCC, one DCC mutation leading to a frameshift, 5 missense variants in DCC, and 2 missense variants in RAD51. All 7 missense variants were predicted to be pathogenic by one or both algorithms. Statistical analysis showed that the frequency of variants predicted to be deleterious was significantly different between patients and controls (p < 0.001 for both RAD51 and DCC). CONCLUSION: Mutations and variants in DCC and RAD51 are strongly associated with CMM, but additional genes causing CMM remain to be discovered.

Early muscle and brain ultrastructural changes in polymerase gamma 1-related encephalomyopathy.

Mutations affecting the mitochondrial DNA-polymerase gamma 1 (POLG1) gene have been shown to cause Alpers-Huttenlocher disease. Ultrastructural data on brain and muscle tissue are rare. We report on ultrastructural changes in brain and muscle tissue of two sisters who were compound heterozygous for the c.2243G>C and c.1879C>T POLG1 mutations. Patient 1 (16 years) presented with epilepsia partialis continua that did not respond to antiepileptic treatment. Neuroimaging showed right occipital and bithalamic changes. Light microscopy from a brain biopsy performed after 3 weeks suggested chronic encephalitis showing astro- and microgliosis as well as perivascular CD8-positive T-cells. However, immunosuppressive therapy failed to improve her condition. When her 17-year-old sister (patient 2) also developed epilepsy, an intensified search for metabolic diseases led to the diagnosis. On electron microscopy mitochondrial abnormalities mainly affecting neurons were detected in the brain biopsy of patient 1, including an increase in number and size, structural changes and globoid inclusions. In patient 2, light and electron microscopy on a muscle biopsy confirmed a mitochondrial myopathy, also revealing an increase in mitochondrial size and number, as well as globoid inclusions. Neurons may be the primary target of mitochondrial dysfunction in brains of patients with Alpers disease related to POLG1 mutations. During early disease stages, brain histopathology may be misleading, showing reactive inflammatory changes.

Primary pulmonary hypertension, congenital heart defect, central nervous system malformations, hypo- and aplastic toes: another case of Yunis-Varón syndrome or report of a new entity.

Here we describe a patient with a new malformation syndrome which shows similarities with Yunis-Varon syndrome (YVS). Prenatal presentation included polyhydramnios, increased nuchal translucency, and bilateral hydrothoraces requiring pigtail insertion. Postnatal presentation revealed primary pulmonary hypertension (PPH), persistent hydrothoraces, one atrial and two ventricular septal defects, hypoplasia of the corpus callosum and cerebellar vermis, dilated interhemispheric ventricles, severe developmental delay with general muscular hypotonia, retinal anomalies, sparse scalp hair, sparse eyebrows and eyelashes, hypo- and aplastic nails, low-set dysplastic ears, loose nuchal skin, hypo- and aplastic distal phalanges of the toes as well as postnatal failure to thrive. High resolution molecular karyotyping in the patient did not reveal any causative chromosomal aberration. Since one patient with YVS and PPH has been previously reported, we assume a similar pathogenic pathway. However, molecular confirmation of the clinical diagnosis is not yet possible. It remains uncertain if the presented syndrome can be classified as YVS with PPH or if it constitutes a new YVS like entity.

Digital necroses and vascular thrombosis in severe spinal muscular atrophy.

Infantile spinal muscular atrophy (SMA) caused by homozygous SMN1 gene deletions/mutations is characterized by neuronal loss and axonopathy of motor neurons. We report two unrelated patients with severe SMA type I who had only one SMN2 copy and developed ulcerations and necroses of the fingers and toes. Sural nerve biopsy was normal in patient 1, whose affected skin displayed necroses and thrombotic occlusions of small vessels. Corresponding to a mouse model and other patients with similar findings, we believe that severe survival motor neuron (SMN) deficiency may present as vasculopathy.

Novel CCM1, CCM2, and CCM3 mutations in patients with cerebral cavernous malformations: in-frame deletion in CCM2 prevents formation of a CCM1/CCM2/CCM3 protein complex.

Cerebral cavernous malformations (CCM) are prevalent cerebrovascular lesions predisposing to chronic headaches, epilepsy, and hemorrhagic stroke. Using a combination of direct sequencing and MLPA analyses, we identified 15 novel and eight previously published CCM1 (KRIT1), CCM2, and CCM3 (PDCD10) mutations. The mutation detection rate was >90% for familial cases and >60% for isolated cases with multiple malformations. Splice site mutations constituted almost 20% of all CCM mutations identified. One of these proved to be a de novo mutation of the most 3' acceptor splice site of the CCM1 gene resulting in retention of intron 19. A further mutation affected the 3' splice site of CCM2 intron 2 leading to cryptic splice site utilization in both CCM2 and its transcript variant lacking exon 2. With the exception of one in-frame deletion of CCM2 exon 2, which corresponds to the naturally occurring splice variant of CCM2 on the RNA level and is predicted to result in the omission of 58 amino acids (CCM2:p.P11_K68del), all mutations lead to the introduction of premature stop codons. To gain insight into the likely mechanisms underlying the only known CCM2 in-frame deletion, we analyzed the functional consequences of loss of CCM2 exon 2. The CCM2:p.P11_K68del protein could be expressed in cell culture and complexed with CCM3. However, its ability to interact with CCM1 and to form a CCM1/CCM2/CCM3 complex was lost. These data are in agreement with a loss-of-function mechanism for CCM mutations, uncover an N-terminal CCM2 domain required for CCM1 binding, and demonstrate full-length CCM2 as the essential core protein in the CCM1/CCM2/CCM3 complex.