Livia Garavelli

Heterozygous missense mutations in SMARCA2 cause Nicolaides-Baraitser syndrome

Nicolaides-Baraitser syndrome (NBS) is characterized by sparse hair, distinctive facial morphology, distal-limb anomalies and intellectual disability. We sequenced the exomes of ten individuals with NBS and identified heterozygous variants in SMARCA2 in eight of them. Extended molecular screening identified nonsynonymous SMARCA2 mutations in 36 of 44 individuals with NBS; these mutations were confirmed to be de novo when parental samples were available. SMARCA2 encodes the core catalytic unit of the SWI/SNF ATP-dependent chromatin remodeling complex that is involved in the regulation of gene transcription. The mutations cluster within sequences that encode ultra-conserved motifs in the catalytic ATPase region of the protein. These alterations likely do not impair SWI/SNF complex assembly but may be associated with disrupted ATPase activity. The identification of SMARCA2 mutations in humans provides insight into the function of the Snf2 helicase family.

Mowat-Wilson syndrome

Mowat-Wilson syndrome (MWS) is a multiple congenital anomaly syndrome characterized by a distinct facial phenotype (high forehead, frontal bossing, large eyebrows, medially flaring and sparse in the middle part, hypertelorism, deep set but large eyes, large and uplifted ear lobes, with a central depression, saddle nose with prominent rounded nasal tip, prominent columella, open mouth, with M-shaped upper lip, frequent smiling, and a prominent but narrow and triangular pointed chin), moderate-to-severe intellectual deficiency, epilepsy and variable congenital malformations including Hirschsprung disease (HSCR), genitourinary anomalies (in particular hypospadias in males), congenital heart defects, agenesis of the corpus callosum and eye anomalies. The prevalence of MWS is currently unknown, but 171 patients have been reported so far. It seems probable that MWS is under-diagnosed, particularly in patients without HSCR. MWS is caused by heterozygous mutations or deletions in the Zinc finger E-box-binding homeobox 2 gene, ZEB2, previously called ZFHX1B (SIP1). To date, over 100 deletions/mutations have been reported in patients with a typical phenotype; they are frequently whole gene deletions or truncating mutations, suggesting that haploinsufficiency is the main pathological mechanism. Studies of genotype-phenotype analysis show that facial gestalt and delayed psychomotor development are constant clinical features, while the frequent and severe congenital malformations are variable. In a small number of patients, unusual mutations can lead to an atypical phenotype. The facial phenotype is particularly important for the initial clinical diagnosis and provides the hallmark warranting ZEB2 mutational analysis, even in the absence of HSCR. The majority of MWS cases reported so far were sporadic, therefore the recurrence risk is low. Nevertheless, rare cases of sibling recurrence have been observed. Congenital malformations and seizures require precocious clinical investigation with intervention of several specialists (including neonatologists and pediatricians). Psychomotor development is delayed in all patients, therefore rehabilitation (physical therapy, psychomotor and speech therapy) should be started as soon as possible.

Congenital heart defects: 15 years of experience of the Emilia-Romagna Registry (Italy)

Objectives: Collection and assessment of data from the Emilia-Romagna Region on the occurrence of congenital heart defects in order to identify an homogeneous group of patients for further aetiologic and genetic studies. Materials and methods: The present study is based on 1549 stillborn and live born babies affected by congenital heart defect out of 330,017 consecutive births (4.7 per 1000). Results: The frequency and type of congenital heart defects have been identified together with the sex ratio, associated extracardiac anomalies, chromosomal anomalies and the risk of precurrence in relatives. The impact of prenatal diagnosis on prevalence was low during the study period. Conclusions: The study has provided epidemiological data for public health surveillance of congenital heart defects in the Emilia-Romagna region. The creation of a system for the nationwide recording of congenital heart defects designed with regard to the sources of ascertainment, the diagnostic criteria, and the system of classification is emphasised.

Mowat–Wilson syndrome: Facial phenotype changing with age: Study of 19 Italian patients and review of the literature

Mowat–Wilson syndrome (MWS; OMIM #235730) is a genetic condition caused by heterozygous mutations or deletions of the ZEB2 gene, and characterized by typical face, moderate‐to‐severe mental retardation, epilepsy, Hirschsprung disease, and multiple congenital anomalies, including genital anomalies (particularly hypospadias in males), congenital heart defects, agenesis of the corpus callosum, and eye defects. Since the first delineation by Mowat et al. [Mowat et al. ( 1998 ); J Med Genet 35:617–623], ∼179 patients with ZEB2 mutations, deletions or cytogenetic abnormalities have been reported primarily from Europe, Australia and the United States. Genetic defects include chromosome 2q21–q23 microdeletions (or different chromosome rearrangements) in few patients, and ZEB2 mutations in most. We report on clinical and genetic data from 19 Italian patients, diagnosed within the last 5 years, including six previously published, and compare them with patients already reported. The main purpose of this review is to underline a highly consistent phenotype and to highlight the phenotypic evolution occurring with age, particularly of the facial characteristics. The prevalence of MWS is likely to be underestimated. Knowledge of the phenotypic spectrum of MWS and of its changing phenotype with age can improve the detection rate of this condition.

Phenotypic and molecular characterisation of the Aarskog–Scott syndrome: a survey of the clinical variability in light of FGD1 mutation analysis in 46 patients

Faciogenital dysplasia or Aarskog–Scott syndrome (AAS) is a genetically heterogeneous developmental disorder. The X-linked form of AAS has been ascribed to mutations in the FGD1 gene. However, although AAS may be considered as a relatively frequent clinical diagnosis, mutations have been established in few patients. Genetic heterogeneity and the clinical overlap with a number of other syndromes might explain this discrepancy. In this study, we have conducted a single-strand conformation polymorphism (SSCP) analysis of the entire coding region of FGD1 in 46 AAS patients and identified eight novel mutations, including one insertion, four deletions and three missense mutations (19.56% detection rate). One mutation (528insC) was found in two independent families. The mutations are scattered all along the coding sequence. Phenotypically, all affected males present with the characteristic AAS phenotype. FGD1 mutations were not associated with severe mental retardation. However, neuropsychiatric disorders, mainly behavioural and learning problems in childhood, were observed in five out of 12 mutated individuals. The current study provides further evidence that mutations of FGD1 may cause AAS and expands the spectrum of disease-causing mutations. The importance of considering the neuropsychological phenotype of AAS patients is discussed.

PRKACB and Carney Complex

The authors report that a gain of function in the catalytic subunit beta of the cyclic AMP–dependent protein kinase (protein kinase A), resulting from the presence of four copies of PRKACB (instead of the normal two), may lead to a Carney complex phenotype.

Mutation spectrum of MLL2 in a cohort of kabuki syndrome patients

BackgroundKabuki syndrome (Niikawa-Kuroki syndrome) is a rare, multiple congenital anomalies/mental retardation syndrome characterized by a peculiar face, short stature, skeletal, visceral and dermatoglyphic abnormalities, cardiac anomalies, and immunological defects. Recently mutations in the histone methyl transferase MLL2 gene have been identified as its underlying cause.MethodsGenomic DNAs were extracted from 62 index patients clinically diagnosed as affected by Kabuki syndrome. Sanger sequencing was performed to analyze the whole coding region of the MLL2 gene including intron-exon junctions. The putative causal and possible functional effect of each nucleotide variant identified was estimated by in silico prediction tools.ResultsWe identified 45 patients with MLL2 nucleotide variants. 38 out of the 42 variants were never described before. Consistently with previous reports, the majority are nonsense or frameshift mutations predicted to generate a truncated polypeptide. We also identified 3 indel, 7 missense and 3 splice site.ConclusionsThis study emphasizes the relevance of mutational screening of the MLL2 gene among patients diagnosed with Kabuki syndrome. The identification of a large spectrum of MLL2 mutations possibly offers the opportunity to improve the actual knowledge on the clinical basis of this multiple congenital anomalies/mental retardation syndrome, design functional studies to understand the molecular mechanisms underlying this disease, establish genotype-phenotype correlations and improve clinical management.

Loss-of-function mutations in PTPN11 cause metachondromatosis, but not Ollier disease or Maffucci syndrome.

Metachondromatosis (MC) is a rare, autosomal dominant, incompletely penetrant combined exostosis and enchondromatosis tumor syndrome. MC is clinically distinct from other multiple exostosis or multiple enchondromatosis syndromes and is unlinked to EXT1 and EXT2, the genes responsible for autosomal dominant multiple osteochondromas (MO). To identify a gene for MC, we performed linkage analysis with high-density SNP arrays in a single family, used a targeted array to capture exons and promoter sequences from the linked interval in 16 participants from 11 MC families, and sequenced the captured DNA using high-throughput parallel sequencing technologies. DNA capture and parallel sequencing identified heterozygous putative loss-of-function mutations in PTPN11 in 4 of the 11 families. Sanger sequence analysis of PTPN11 coding regions in a total of 17 MC families identified mutations in 10 of them (5 frameshift, 2 nonsense, and 3 splice-site mutations). Copy number analysis of sequencing reads from a second targeted capture that included the entire PTPN11 gene identified an additional family with a 15 kb deletion spanning exon 7 of PTPN11. Microdissected MC lesions from two patients with PTPN11 mutations demonstrated loss-of-heterozygosity for the wild-type allele. We next sequenced PTPN11 in DNA samples from 54 patients with the multiple enchondromatosis disorders Ollier disease or Maffucci syndrome, but found no coding sequence PTPN11 mutations. We conclude that heterozygous loss-of-function mutations in PTPN11 are a frequent cause of MC, that lesions in patients with MC appear to arise following a “second hit,” that MC may be locus heterogeneous since 1 familial and 5 sporadically occurring cases lacked obvious disease-causing PTPN11 mutations, and that PTPN11 mutations are not a common cause of Ollier disease or Maffucci syndrome.

Hirschsprung disease, mental retardation, characteristic facial features, and mutation in the gene ZFHX1B (SIP1): Confirmation of the Mowat-Wilson syndrome

We read with interest the article ‘‘Hirschsprung disease, microcephaly, mental retardation, and characteristic facial features: delineation of a new syndrome and identification of a locus at chromosome 2q22–q23’’ [Mowat et al., 1998]. The same distinctive face was reported by Kääriäinen et al. [2001] in four out of five patients in our opinion and by Zweier et al. [2002]. Recently, mutations in the zinc finger homeobox 1 B gene, ZFHX1B (SIP1), were identified in patients with and without Hirschsprung disease (HSCR), multiple congenital anomalies, mental retardation (MCA/MR), and the same facial gestalt [Zweier et al., 2002]. The latter authors propose the name, Mowat-Wilson syndrome for this entity. It has been demonstrated that patients 1, 2, and 3 of Mowat et al. [1998] also have a heterozygous mutation in the gene ZFHX1B (SIP1) [Cacheux et al., 2001]. In other patients, the correlation with the facial gestalt has not yet been clearly established [Amiel et al., 2001; Wakamatsu et al., 2001; Yamada et al., 2001]. Wehave recently seen aboywithHSCRwith the same phenotype as the cases described by Mowat et al. and having a mutation in the gene ZFHX1B (SIP1). The boy is the first of two children of healthy, nonconsanguineous parents (34-year-old mother and 34-year-old father). The mother’s sister had Noonan syndrome. Family history was otherwise unremarkable, none of the familymembers showedHSCR.Prenatalultrasound at 15 weeks gestation showed bilateral mild dilatation of the renal pelvis. There were poor fetal movements. Hewasbornat 40weeksgestation. Spontaneousvaginal vertex delivery occurred with a birth weight of 3,750 g, length of 51 cm, and head circumference of 34 cm. APGAR scores were 9 and 10 at one and five minutes, respectively. He was transferred from the Neonatal Service to pediatric surgery at the age of two days and underwent surgery for HSCR at the age of 50 days. He had hypotonia and global developmental delay, with involuntary nodding of the head and exaggerated reaction to acoustic stimulus. At the age of 17 months, he developed recurrent seizures, which responded to anticonvulsants, and partial epilepsy complex was diagnosed. On examination at the age of 18 months, his head circumference was 46 cm (10th centile), height was 83.5 cm (50th–75th centile), and weight was 10.16 kg (10th centile) He had fine and blond hair, high forehead, frontal bossing, prominent supraorbital ridges, deep set eyes, small nose with a bulbous tip, mouth held open, pointed chin, posterior angulation of ears, uplifted ear lobes, fetal finger pads, deep palmar and plantar creases, long halluces, hypospadias, and undescended right testis (Figs. 1 and 2). Dermatoglyphics showed 8/10 ulnar loops. Investigations including lactate, ammonium, QFQ banded karyotype at 650 band resolution, molecular analyses for fragile X syndrome, and flourescence in situ hybridization (FISH) for chromosome 8 mosaicism were all normal. Skeletal survey showed Wormian bones; hypoplasia of the distal phalanges of the fingers and hypoplasia of the middle and distal phalanges of the 2nd, 3rd, 4th, and 5th toes; and long and hypertrophic first ray of the feet. The patellae were normal. Cranial computed tomography (CT) demonstrated greater representation of subarachnoid spaces in frontal side compared to the remaining sites of the head, and magnetic resonance imaging (MRI) of the brain showed mild symmetrical accentuation of the apex of temporal horns bilaterally.Abdominal ultrasonographic evaluation was normal. Direct sequencing of ZFHX1B revealed a de novo heterozygous frameshift mutation nt901delC in exon 7, resulting in a stop codon after 36 aa. The truncated protein is missing part of the N-terminal zinc finger domain, the Smad binding domain, the homeodomain, and the C-terminal zinc finger domain (Fig. 3). The analysis was performed as described by Zweier et al. [2002]. *Correspondence to: Dr. Livia Garavelli, Divisione di Pediatria, Ambulatorio di Genetica Clinica, Arcispedale S. Maria NuovaAziendaOspedaliera-Viale Risorgimento, 80 42100 Reggio Emilia, Italy. E-mail: garavelli.livia@asmn.re.it

Loss of the BMP Antagonist, SMOC-1, Causes Ophthalmo-Acromelic (Waardenburg Anophthalmia) Syndrome in Humans and Mice

Ophthalmo-acromelic syndrome (OAS), also known as Waardenburg Anophthalmia syndrome, is defined by the combination of eye malformations, most commonly bilateral anophthalmia, with post-axial oligosyndactyly. Homozygosity mapping and subsequent targeted mutation analysis of a locus on 14q24.2 identified homozygous mutations in SMOC1 (SPARC-related modular calcium binding 1) in eight unrelated families. Four of these mutations are nonsense, two frame-shift, and two missense. The missense mutations are both in the second Thyroglobulin Type-1 (Tg1) domain of the protein. The orthologous gene in the mouse, Smoc1, shows site- and stage-specific expression during eye, limb, craniofacial, and somite development. We also report a targeted pre-conditional gene-trap mutation of Smoc1 (Smoc1tm1a) that reduces mRNA to ∼10% of wild-type levels. This gene-trap results in highly penetrant hindlimb post-axial oligosyndactyly in homozygous mutant animals (Smoc1tm1a/tm1a). Eye malformations, most commonly coloboma, and cleft palate occur in a significant proportion of Smoc1tm1a/tm1a embryos and pups. Thus partial loss of Smoc-1 results in a convincing phenocopy of the human disease. SMOC-1 is one of the two mammalian paralogs of Drosophila Pentagone, an inhibitor of decapentaplegic. The orthologous gene in Xenopus laevis, Smoc-1, also functions as a Bone Morphogenic Protein (BMP) antagonist in early embryogenesis. Loss of BMP antagonism during mammalian development provides a plausible explanation for both the limb and eye phenotype in humans and mice.