Tracheal agenesis versus tracheal atresia: anatomical conditions, pathomechanisms and causes with a possible link to a novel MAPK11 variant in one case.

BACKGROUND: In this study we aimed to describe the morphological and pathogenetic differences between tracheal agenesis and tracheal atresia, which are not clearly distinguished from each other in the literature, and to contribute thereby to the understanding and management of these conditions. Both tracheal agenesis and tracheal atresia represent rare disorders of still unknown aetiology that cannot be detected by prenatal ultrasound. If the affected foetuses survive until birth these conditions result in respiratory failure and in futile attempts to rescue the infant's life. RESULTS: Autopsies and genetic analyses, including singleton or trio exome sequencing, were performed on five neonates/foetuses with tracheal agenesis and three foetuses with tracheal atresia. Tracheal agenesis was characterized by absence of the sublaryngeal trachea and presence of a bronchooesophageal fistula and by pulmonary isomerism and occurred as an isolated malformation complex or as part of a VACTERL association. Special findings were an additional so-called 'pig bronchus' and a first case of tracheal agenesis with sirenomelia. Tracheal atresia presenting with partial obliteration of its lumen and persistence of a fibromuscular streak resulted in CHAOS. This condition was associated with normal lung lobulation and single, non-VACTERL type malformations. Trio ES revealed a novel variant of MAPK11 in one tracheal agenesis case. Its involvement in tracheooesophageal malformation is herein discussed, but remains hypothetical. CONCLUSION: Tracheal agenesis and tracheal atresia represent different disease entities in terms of morphology, pathogenesis and accompanying anomalies due to a primary developmental and secondary disruptive possibly vascular disturbance, respectively.

Brain malformations in diprosopia observed in clinical cases, museum specimens and artistic representations.

BACKGROUND: Diprosopus is a rare malformation of still unclear aetiology. It describes a laterally double faced monocephalic and single-trunk individual and has to be distinguished from the variant Janus type diprosopus. RESULTS: We examined seven double-faced foetuses, five showing true diprosopus, and one each presenting as monocephalic Janiceps and parasitic conjoined twins. Four of the foetuses presented with (cranio)rachischisis, and two had secondary hydrocephaly. Three foetuses showed cerebral duplication with concordant holoprosencephaly, Dandy-Walker cyst and/or intracranial anterior encephalocele. In the Janiceps twins, cerebral duplication was accompanied by cerebral di-symmetry. In the parasitic twins the cyclopic facial aspects were suggestive of concordant holoprosencephaly. In one of the true diprosopus cases, pregnancy was achieved after intracytoplasmic sperm injection. Whole-exome sequencing, perfomed in one case, did not reveal any possible causative variants.The comparison of our double-faced foetuses to corresponding artistic representations from the Tlatilco culture allowed retrospective assignment of hairstyles to brain malformations. CONCLUSION: Brain malformations in patients with diprosopus may not be regarded as an independent event but rather as a sequel closely related to the duplication of the notochord and neural plate and as a consequence of the cerebral and associated craniospinal structural instabilities.

History and highlights of the teratological collection in the Narrenturm, Vienna (Austria).

The collection of the Narrenturm in Vienna houses and maintains more than 50,000 objects including approximately 1200 teratological specimens; making it one of the biggest collections of specimens from human origin in Europe. The existence of this magnificent collection-representing an important resource for dysmorphology research, mostly awaiting contemporary diagnoses-is not widely known in the scientific community. Here, we show that the Narrenturm harbors a wealth of specimens with (exceptionally) rare congenital anomalies. These museums can be seen as physical repositories of human malformation, covering hundreds of years of dedicated collecting and preserving, thereby creating unique settings that can be used to expand our knowledge of developmental conditions that have to be preserved for future generations of scientists.

Prenatal phenotype of PNKP-related primary microcephaly associated with variants affecting both the FHA and phosphatase domain.

Biallelic PNKP variants cause heterogeneous disorders ranging from neurodevelopmental disorder with microcephaly/seizures to adult-onset Charcot-Marie-Tooth disease. To date, only postnatal descriptions exist. We present the first prenatal diagnosis of PNKP-related primary microcephaly. Pathological examination of a male fetus in the 18th gestational week revealed micrencephaly with extracerebral malformations and thus presumed syndromic microcephaly. A recessive disorder was suspected because of previous pregnancy termination for similar abnormalities. Prenatal trio-exome sequencing identified compound heterozygosity for the PNKP variants c.498G>A, p.[(=),0?] and c.302C>T, p.(Pro101Leu). Segregation confirmed both variants in the sister fetus. Through RNA analyses, we characterized exon 4 skipping affecting the PNKP forkhead-associated (FHA) and phosphatase domains (p.Leu67_Lys166del) as the predominant effect of the paternal c.498G>A variant. We retrospectively investigated two unrelated individuals diagnosed with biallelic PNKP-variants to compare prenatal/postnatal phenotypes. Both carry the splice donor variant c.1029+2T>C in trans with a variant in the FHA domain (c.311T>C, p.(Leu104Pro); c.151G>C, p.(Val51Leu)). RNA-seq showed complex splicing for c.1029+2T>C and c.151G>C. Structural modeling revealed significant clustering of missense variants in the FHA domain with variants generating structural damage. Our clinical description extends the PNKP-continuum to the prenatal stage. Investigating possible PNKP-variant effects using RNA and structural modeling, we highlight the mutational complexity and exemplify a PNKP-variant characterization framework.

Compound Heterozygous Frameshift Mutations in MESD Cause a Lethal Syndrome Suggestive of Osteogenesis Imperfecta Type XX.

Multiple genes are known to be associated with osteogenesis imperfecta (OI), a phenotypically and genetically heterogenous bone disorder, marked predominantly by low bone mineral density and increased risk of fractures. Recently, mutations affecting MESD, which encodes for a chaperone required for trafficking of the low-density lipoprotein receptors LRP5 and LRP6 in the endoplasmic reticulum, were described to cause autosomal-recessive OI XX in homozygous children. In the present study, whole-exome sequencing of three stillbirths in one family was performed to evaluate the presence of a hereditary disorder. To further characterize the skeletal phenotype, fetal autopsy, bone histology, and quantitative backscattered electron imaging (qBEI) were performed, and the results were compared with those from an age-matched control with regular skeletal phenotype. In each of the affected individuals, compound heterozygous mutations in MESD exon 2 and exon 3 were detected. Based on the skeletal phenotype, which was characterized by multiple intrauterine fractures and severe skeletal deformity, OI XX was diagnosed in these individuals. Histological evaluation of MESD specimens revealed an impaired osseous development with an altered osteocyte morphology and reduced canalicular connectivity. Moreover, analysis of bone mineral density distribution by qBEI indicated an impaired and more heterogeneous matrix mineralization in individuals with MESD mutations than in controls. In contrast to the previously reported phenotypes of individuals with OI XX, the more severe phenotype in the present study is likely explained by a mutation in exon 2, located within the chaperone domain of MESD, that leads to a complete loss of function, which indicates the relevance of MESD in early skeletal development. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..

Smith-Lemli-Opitz syndrome - Fetal phenotypes with special reference to the syndrome-specific internal malformation pattern.

BACKGROUND: Autosomal-recessive SLOS is caused by mutations in the DHCR7 gene. It is defined as a highly variable complex of microcephaly with intellectual disability, characteristic facies, hypospadias, and polysyndactyly. Syndrome diagnosis is often missed at prenatal ultrasound and fetal autopsy METHODS: We performed autopsies and DHCR7 gene analyses in eight fetuses suspected of having SLOS and measured cholesterol values in long-term formalin-fixed tissues of an additional museum exhibit RESULTS: Five of the nine fetuses presented classical features of SLOS, including four cases with atrial/atrioventricular septal defects and renal anomalies, and one with additional bilateral renal agenesis and a Dandy-Walker cyst. These cases allowed for diagnosis at autopsy and subsequent SLOS diagnosis in two siblings. Two fetuses were mildly affected and two fetuses showed additional holoprosencephaly. These four cases and the exhibit had escaped diagnosis at autopsy. The case with bilateral renal agenesis presented a novel combination of a null allele and a putative C-terminus missense mutation in the DHCR7 gene CONCLUSIONS: In view of the discrepancy between the prevalence of SLOS among newborns and the carrier frequency of a heterozygous DHCR7 gene mutation, the syndrome-specific internal malformation pattern may be helpful not to miss SLOS diagnosis in fetuses at prenatal ultrasound and fetal autopsy.

Distal Arthrogryposis with Impaired Proprioception and Touch: Description of an Early Phenotype in a Boy with Compound Heterozygosity of PIEZO2 Mutations and Review of the Literature.

The recessive PIEZO2-associated disease, distal arthrogryposis with impaired proprioception and touch (DAIPT), is characterized by hypotonia, perinatal respiratory distress, significantly delayed motor milestones, and progressive symptoms of distal arthrogryposis and scoliosis. Here, we describe the youngest patient with DAIPT to date, who, at the age of 3.5 years, did not show a single clinical sign of distal arthrogryposis or contractures, but had a history of bilateral clubfoot operations. On the contrary, he presented with some features, not described thus far, such as syringohydromyelia, a small cyst of the spinal cord, moderate microcephaly with premature closure of anterior fontanelle, and spontaneous unilateral patella dislocation at the age of 32 months. Using whole exome sequencing, we identified 2 new different loss-of-function mutations in the PIEZO2 gene in our patient. We also review the phenotypes of all 16 previously published patients with DAIPT, summarize the distinctive clinical features of this rare genetic disorder, and recommend that DAIPT be included in the differential diagnosis of floppy infant. PIEZO2 is a unique ion channel that converts mechanical impulses into cellular signals and is involved in various mechanotransduction pathways. In addition to DAIPT, mutations in PIEZO2 have been described to cause 3 more distinct phenotypes of distal arthrogryposis, which are dominant and associated with gain-of-function mutations. On the contrary, recessive DAIPT is associated with loss-of-function PIEZO2 mutations.

Piepkorn type of osteochondrodysplasia: Defining the severe end of FLNB-related skeletal disorders in three fetuses and a 106-year-old exhibit.

The Piepkorn type of lethal osteochondrodysplasia (POCD) is a rare and lethal dwarfing condition. Four cases have been reported to date. The characteristic features are distinctly shortened "flipper-like" limbs, polysyndactyly, excessive underossification, especially of the limb bones and vertebrae, and large (giant) chondrocytes in the cartilaginous bone primordia. These characteristics allowed the diagnosis of Piepkorn type of osteochondrodysplasia in four new cases, three fetuses of 15 to 22 weeks and one 106-year-old museum exhibit. Piepkorn type of osteochondrodysplasia has been assigned to the giant cell chondrodysplasias such as atelosteogenesis type 1 (AO1) and boomerang dysplasia (BD). Analysis of the Filamin B gene in 3p14.3, which is associated with these disorders, allowed the identification of the first FLNB mutations in Piepkorn type of osteochondrodysplasia. The heterozygous missense mutations, found in the three fetuses, were located in exons 28 and 29, encoding the immunoglobulin-like repeat region R15, one of three mutational hot spots in dominant FLNB-related skeletal disorders. Direct preparations and alcian blue staining revealed single upper and lower arm and leg bone primordia, preaxial oligodactyly, and polysyndactyly with complete fusion and doubling of the middle and end phalanges II-V to produce eight distal finger rays. Considering the unique clinical features and the extent of underossification, Piepkorn type of osteochondrodysplasia can be regarded as a distinct entity within the AO1-BD-POCD continuum.

Fetal Pathology of Neural Tube Defects - An Overview of 68 Cases.

INTRODUCTION: The prevalence of neural tube defects worldwide is 1 - 2 per 1000 neonates. Neural tube defects result from a disturbance of neurulation in the 3rd or 4th week of development and thus represent the earliest manifestation of organ malformation. Neural tube defects (NTD) are classified into cranial dysraphism leading to anencephaly or meningoencephalocele and spinal dysraphism with or without meningomyelocele. In isolated form they have multifactorial causes, and the empirical risk of recurrence in Central Europe is 2%. As associated malformations they tend to occur sporadically, and in monogenic syndromes they follow Mendelian inheritance patterns with a high risk of recurrence. PATIENTS: Autopsies were performed on 68 fetuses following a prenatal diagnosis of NTD and induced abortion. RESULTS: The incidence of NTDs in our autopsied fetuses was 8% and 11% in fetuses with malformations. The percentage of fetuses with anencephaly, encephalocele or spina bifida was 24, 18, and 60%*, respectively. Analysis of the sex distribution showed a female preponderance in cranial dysraphisms but the sex distribution of spina bifida cases was equal. The extent and localization of NTDs varied, with lumbosacral cases clearly predominating. The proportion of isolated, associated and syndromic neural tube defects was 56, 23.5 and 20.6% respectively. In the majority of syndromes, the neural tube defect represented a not previously observed syndromic feature. CONCLUSION: The high proportion of NTDs with monogenic background underlines the importance of a syndrome oriented fetal pathology. At the very least it requires a thourough photographic and radiographic documentation of the fetal phenotype to enable the genetic counsellor to identify a syndromic disorder. This is necessary to determine the risk of recurrence, arrange confirming mutation analyses and offer targeted prenatal diagnosis in subsequent pregnancies.

Mutations in TGDS associated with additional malformations of the middle fingers and halluces: Atypical Catel-Manzke syndrome in a fetus.

Pierre-Robin sequence, radial deviation, and ulnar clinodactyly of the index fingers due to an additional phalangeal bone, as well as heart defects are the key features of Catel-Manzke syndrome. Although mutations in TGDS were identified as the cause of this disorder, the pathogenetic mechanism remains unknown. Here, we report on a fetus with severe heart defect, nuchal edema, talipes, Pierre-Robin sequence, and bilateral deviation and clinodactyly of the index and middle fingers. Pregnancy was terminated at the 22nd week of gestation. Postmortem radiographs showed hypoplasia and V-shaped displacement of the second and third proximal phalanges of both hands as well as hypoplasia of the first metatarsals and the phalangeal bones of the halluces. The suggested diagnosis Catel-Manzke syndrome was confirmed by the detection of two compound heterozygous mutations in TGDS: The known variant c.298G>T; p.(Ala100Ser) and the so far undescribed variant c.895G>A; p.(Asp299Asn), located in the predicted substrate binding site of TGDS. This is the first report on the association of mutations in TGDS with additional anomalies of the middle fingers and halluces. We provide a detailed phenotypic characterization of the only fetus with molecularly confirmed Catel-Manzke syndrome, which is relevant for prenatal diagnosis. Our findings widen the phenotype spectrum caused by TGDS mutations and underline the phenotypic overlap with Temtamy preaxial brachydactyly syndrome. This improves our understanding of the prenatal development and the pathogenetic mechanism of Catel-Manzke syndrome.

De Novo Mutations in SON Disrupt RNA Splicing of Genes Essential for Brain Development and Metabolism, Causing an Intellectual-Disability Syndrome.

The overall understanding of the molecular etiologies of intellectual disability (ID) and developmental delay (DD) is increasing as next-generation sequencing technologies identify genetic variants in individuals with such disorders. However, detailed analyses conclusively confirming these variants, as well as the underlying molecular mechanisms explaining the diseases, are often lacking. Here, we report on an ID syndrome caused by de novo heterozygous loss-of-function (LoF) mutations in SON. The syndrome is characterized by ID and/or DD, malformations of the cerebral cortex, epilepsy, vision problems, musculoskeletal abnormalities, and congenital malformations. Knockdown of son in zebrafish resulted in severe malformation of the spine, brain, and eyes. Importantly, analyses of RNA from affected individuals revealed that genes critical for neuronal migration and cortex organization (TUBG1, FLNA, PNKP, WDR62, PSMD3, and HDAC6) and metabolism (PCK2, PFKL, IDH2, ACY1, and ADA) are significantly downregulated because of the accumulation of mis-spliced transcripts resulting from erroneous SON-mediated RNA splicing. Our data highlight SON as a master regulator governing neurodevelopment and demonstrate the importance of SON-mediated RNA splicing in human development.

Dilated, arrhythmogenic cardiomyopathy in emery-dreifuss muscular dystrophy due to the emerin splice-site mutation c.449 + 1G>A.

OBJECTIVE: Cardiac involvement in X-linked Emery-Dreifuss muscular dystrophy (X-EDMD) usually includes arrhythmias but not dilative cardiomyopathy (dCMP). Here, we report an X-EDMD patient with severe dCMP and life-threatening ventricular arrhythmias associated with other phenotypic features unusual for X-EDMD. CASE REPORT: A 46-year-old patient with X-EDMD due to the known splice-site mutation c.449 + 1G>A in the emerin gene experienced palpitations for the first time at the age of 21 years, and a first syncope at the age of 23 years. He was started on phenprocoumon due to atrial fibrillation and systolic dysfunction. At the age of 28 years he received his first pacemaker. Echocardiography at the age of 36 years showed left ventricular dilatation, enlarged atria, myocardial thickening, 28% ejection fraction and diastolic dysfunction. dCMP was suspected. At the age of 38 years, a cardiac resynchronization therapy system was implanted, which was upgraded to an implantable cardioverter defibrillator (ICD) because of ventricular tachycardias (at the age of 42 years). During the following months, the ICD discharged 30 times due to ventricular tachycardias. In May 2013, he required recurrent cardio-pulmonary resuscitation because ventricular fibrillation occurred with no discharge of the ICD. He was listed for heart transplantation. He also had hypothyroidism, liver hemangiomas, thrombopenia, anemia and diverticulosis. CONCLUSIONS: X-EDMD may occur along with dCMP. An ICD may be ineffective for ventricular fibrillation in X-EDMD. X-EDMD may be associated with unusual, atypical phenotypic features.

Hydrocephalus, agenesis of the corpus callosum, and cleft lip/palate represent frequent associations in fetuses with Peters' plus syndrome and B3GALTL mutations. Fetal PPS phenotypes, expanded by Dandy Walker cyst and encephalocele.

OBJECTIVE: Fetal pathology aims to recognize syndromal patterns of anomalies for goal-directed mutation analyses, genetic counseling, and early prenatal diagnosis in consecutive pregnancies. Here, we report on five fetuses with Peters' plus syndrome (PPS) from two distinct families aborted after prenatal ultrasound diagnosis of hydrocephaly. METHOD: We performed fetal autopsies and molecular analyses. RESULTS: Among 44 fetuses with prenatally diagnosed hydrocephaly, four fetuses of 16 to 21 gestational weeks presented with additional cleft lip/palate and/or agenesis of the corpus callosum. Other features were growth retardation, hypertelorism, anomalies of the eyes, in part consistent with Peters' anterior chamber anomalies, mild brachymelia, brachydactyly, and also internal anomalies. Suspected PPS was confirmed by detection of B3GALTL mutation in these four fetuses and in one additional sib fetus, revealing homozygosity for the common c.660 + 1G > A donor splice site mutation in intron 8. CONCLUSIONS: Autosomal-recessive PPS has not yet been diagnosed prenatally. We want to alert ultrasonographers to the diagnosis of this disorder in growth-retarded fetuses with (recurrent) hydrocephaly, agenesis of the corpus callosum, and cleft lip/palate and stress the more severe fetal manifestation, describing a first such case with additional Dandy-Walker cyst and occult meningoencephalocele.

Klinefelter twins presenting with discordant aneuploidies, acardia, forked umbilical cord and with different gonadal sex despite monozygosity.

OBJECTIVE: A higher frequency of twin births in sibships of Klinefelter syndrome patients and also monozygotic or dizygotic twins, themselves being affected by Klinefelter syndrome have been noted repeatedly. To address this issue, we evaluated type and frequency of twinning among Klinefelter fetuses that we had received for autopsy within a 'Prenatal Diagnosis' program. METHOD: We performed fetal autopsies, and genetic analyses on DNA extracted from stained histological slides. RESULTS: Among 41 prenatal diagnoses of a 47, XXY karyotype we observed four twin pairs. One was dizygotic with discordant Klinefelter and Down syndrome. Three twin pairs were monozygotic as concluded from monochorial placentation. In two monozygotic pairs one twin partner was an acardiac monster and in one of these the acardiac twin showed a female gonadal sex and missing Y-chromosomal SRY-sequences as confirmed by polymerase chain reaction. CONCLUSIONS: There is a high rate of twinning and twin reversed arterial perfusion sequence among our Klinefelter fetuses. Forked umbilical cords at the placental insertion site in one case allowed classification as conjoined twins in the sense of a 'funiculopagus'. Anaphase lagging or semidizygosity by second polar body twinning are proposed as explanations for the gonadal sex discordance and the excessive developmental disadvantage in the one acardiac. Problems may arise with regard to non-invasive prenatal diagnosis of aneuploidies in twin pregnancies.

Recurrent Johanson-Blizzard syndrome in a triplet pregnancy complicated by urethral obstruction sequence: a clinical, molecular, and immunohistochemical approach.

We report on a triplet pregnancy of consanguineous parents with one fetus being affected by recurrent Johanson-Blizzard syndrome (JBS). At autopsy in the 35th gestational week, the affected triplet presented with an especially severe and lethal manifestation of the disorder as compared to his elder affected brother and to cases in the literature, thus exemplifying great interfamilial and intrafamilial phenotypic variability. Arhinencephaly and cystic renal dysplasia associated with urethral obstruction sequence were features not described previously in the literature. In addition to the lack of exocrine acini as the characteristic feature of JBS, the pancreas revealed a resorptive inflammatory reaction with infiltration by eosinophilic granulocytes that focally dispersed onto islets of Langerhans, thus favoring a progressive destructive rather than primary dysplastic process and possibly explaining the occurrence of diabetes mellitus in later life. JBS maps to chromosome 15q15-q21.1 and is associated with mutations in the UBR1 gene. Testing the fetus and the affected sibling revealed a homozygous truncating mutation in UBR1. The resulting absence of the UBR1 protein was confirmed by Western blot. Immunohistochemical staining using a commercial anti-UBR1 antibody demonstrated staining, presumably artifactual. This finding suggests that, until an appropriately validated antibody has been identified, this modality should not be utilized for diagnosis or confirmation of this disorder.

Desbuquois dysplasia type I and fetal hydrops due to novel mutations in the CANT1 gene.

We report on three hydropic fetuses of 17, 22 and 25 gestational weeks from three distinct families presenting with Desbuquois dysplasia type 1. All fetuses showed brachymelia and characteristic dysmorphic features. X-ray studies revealed δ-shaped extraphalangeal bones and disease-specific prominence of the lesser trochanter, varying in severity with fetal age. Early lethal manifestation of the disorder was reflected in lung hypoplasia and in early death of similarly affected siblings in cases 1 and 2. All families were German Caucasians by descent. Sequence analysis of the CANT1 gene revealed two frameshift mutations, c.228_229insC and c.277_278delCT, in homozygous and compound heterozygous configuration, respectively, and a homozygously novel missense mutation, c.336C>A (p.D112E), located within a highly conserved region of exon 2. Haplotype analyses by high-resolution single-nucleotide polymorphism array showed that the haplotype associated with c.228_229insC may be traced to a single founder in the German population.

Fetal manifestation of the Fine-Lubinsky syndrome. Brachycephaly, deafness, cataract, microstomia and mental retardation syndrome complicated by Pierre-Robin anomaly and polyhydramnios.

OBJECTIVES: We report on a female fetus of 24 weeks gestational age with Fine-Lubinsky syndrome (FLS), representing the 7th case published so far. METHODS: Prenatal ultrasound was performed at 22+1 weeks of gestation and thorough postmortem examination was made after termination of pregnancy. RESULTS: The diagnosis of FLS in the fetus was based on characteristic features that were already apparent in early prenatal life, such as growth deficiency, brachycephaly, flat face with associated dysmorphic signs, microstomia and cataract, while deafness and mental retardation, which are syndrome-specific functional disorders and evident only postnatally, could not be taken into account. CONCLUSIONS: This case demonstrates the diagnostic problems in fetal syndromology if syndrome-specific features are not yet recognizable and additional complications occur that had not been observed in this disorder.

Severe facial clefts in acrofacial dysostosis: a consequence of prenatal exposure to mycophenolate mofetil?

BACKGROUND: Immunosuppressants are teratogenic in mice, rats, and rabbits and cause prenatal growth restriction in humans. As yet, there has been no proven teratogenicity in humans. CASE: We present a chromosomally normal fetus with severe acrofacial dysostosis and orofacial clefts. These were bilateral transverse and oblique clefts and defects of the midface. In addition, there were preaxial limb anomalies with digitalization of thumbs and internal cardiovascular, gastrointestinal, and urogenital malformations. The mother had been treated with high doses of the immunosuppressant mycophenolate mofetil in early pregnancy for systemic lupus erythematosus. CONCLUSION: Mycophenolate mofetil may have contributed to or even caused acrofacial dysostosis phenotype and extensive clefting.