Deleterious de novo variants of X-linked ZC4H2 in females cause a variable phenotype with neurogenic arthrogryposis multiplex congenita.

Pathogenic variants in the X-linked gene ZC4H2, which encodes a zinc-finger protein, cause an infrequently described syndromic form of arthrogryposis multiplex congenita (AMC) with central and peripheral nervous system involvement. We present genetic and detailed phenotypic information on 23 newly identified families and simplex cases that include 19 affected females from 18 families and 14 affected males from nine families. Of note, the 15 females with deleterious de novo ZC4H2 variants presented with phenotypes ranging from mild to severe, and their clinical features overlapped with those seen in affected males. By contrast, of the nine carrier females with inherited ZC4H2 missense variants that were deleterious in affected male relatives, four were symptomatic. We also compared clinical phenotypes with previously published cases of both sexes and provide an overview on 48 males and 57 females from 42 families. The spectrum of ZC4H2 defects comprises novel and recurrent mostly inherited missense variants in affected males, and de novo splicing, frameshift, nonsense, and partial ZC4H2 deletions in affected females. Pathogenicity of two newly identified missense variants was further supported by studies in zebrafish. We propose ZC4H2 as a good candidate for early genetic testing of males and females with a clinical suspicion of fetal hypo-/akinesia and/or (neurogenic) AMC.

Measurements of C-reactive protein (CRP) and nerve-growth-factor (NGF) concentrations in serum and urine samples of dogs with neurologic disorders.

BACKGROUND: The purpose of this study was to prove the hypothesis that C-reactive protein (CRP) and nerve growth factor (NGF) may be potential biomarkers for lower urinary tract disorders and may be able to distinguish between micturition dysfunctions of different origin in dogs with spinal cord diseases. NGF- and CRP- concentrations were measured in serum and urine samples using specific ELISA-Kits. Results in urine were standardized by urine-creatinine levels. RESULTS: CRP in serum was detectable in 32/76 and in urine samples in 40/76 patients. NGF could be measured in all serum and in 70/76 urine samples. Urinary CRP concentrations were significantly higher in dogs with micturition dysfunction (p = 0.0009) and in dogs with different neurological diseases (p = 0.0020) compared to the control group. However, comparing dogs with spinal cord disorders with and without associated micturition dysfunction no significant difference could be detected for NGF and CRP values in urine or serum samples. Additionally, levels did not decrease significantly, when measured at the time when the dogs regained the ability to urinate properly (urinary NGF p = 0.7962; urinary CRP p = 0.078). Urine samples with bacteria and/or leukocytes had no significant increase in urinary NGF (p = 0.1112) or CRP (p = 0.0534) concentrations, but higher CRP-levels in urine from dogs with cystitis were found compared to dogs without signs of cystitis. CONCLUSIONS: From these data we conclude that neither CRP nor NGF in urine or serum can be considered as reliable biomarkers for micturition disorders in dogs with spinal cord disorders in a clinical setting, but their production might be part of the pathogenesis of such disorders. Significantly higher levels of CRP could be found in the urine of dogs with micturition dysfunctions compared to control dogs. This phenomenon could potentially be explained by unspecific extrahepatic CRP production by smooth muscle cells in the dilated bladder.

A familial GLI2 deletion (2q14.2) not associated with the holoprosencephaly syndrome phenotype.

Molecular alterations of the GLI2 gene in 2q14.2 are associated with features from the holoprosencephaly spectrum. However, the phenotype is extremely variable, ranging from unaffected mutation heterozygotes to isolated or combined pituitary hormone deficiency, and to patients with a phenotype that overlaps with holoprosencephaly, including abnormal pituitary gland formation/function, craniofacial dysmorphisms, branchial arch anomalies, and polydactyly. Although many point mutations within the GLI2 gene have been identified, large (sub) microscopic deletions affecting 2q14.2 are rare. We report on a family with a 4.3 Mb deletion in 2q14 affecting GLI2 without any dysmorphologic features belonging to the holoprosencephaly spectrum. This family confirms the incomplete penetrance of genomic disturbances affecting the GLI2 gene. However, the family presented here is unique as none of the three identified individuals with a GLI2 deletion showed any typical signs of holoprosencephaly, whereas all patients reported so far were referred for genetic testing because at least one member exhibited holoprosencephaly and related features.

Duplications of BHLHA9 are associated with ectrodactyly and tibia hemimelia inherited in non-Mendelian fashion.

BACKGROUND: Split-hand/foot malformation (SHFM)-also known as ectrodactyly-is a congenital disorder characterised by severe malformations of the distal limbs affecting the central rays of hands and/or feet. A distinct entity termed SHFLD presents with SHFM and long bone deficiency. Mouse models suggest that a defect of the central apical ectodermal ridge leads to the phenotype. Although six different loci/mutations (SHFM1-6) have been associated with SHFM, the underlying cause in a large number of cases is still unresolved. METHODS: High resolution array comparative genomic hybridisation (CGH) was performed in patients with SHFLD to detect copy number changes. Candidate genes were further evaluated for expression and function during limb development by whole mount in situ hybridisation and morpholino knock-down experiments. RESULTS: Array CGH showed microduplications on chromosome 17p13.3, a locus previously associated with SHFLD. Detailed analysis of 17 families revealed that this copy number variation serves as a susceptibility factor for a highly variable phenotype with reduced penetrance, particularly in females. Compared to other known causes for SHFLD 17p duplications appear to be the most frequent cause of SHFLD. A ~11.8 kb minimal critical region was identified encompassing a single gene, BHLHA9, a putative basic loop helix transcription factor. Whole mount in situ hybridisation showed expression restricted to the limb bud mesenchyme underlying the apical ectodermal ridge in mouse and zebrafish embryos. Knock down of bhlha9 in zebrafish resulted in shortening of the pectoral fins. CONCLUSIONS: Genomic duplications encompassing BHLHA9 are associated with SHFLD and non-Mendelian inheritance characterised by a high degree of non-penetrance with sex bias. Knock-down of bhlha9 in zebrafish causes severe reduction defects of the pectoral fin, indicating a role for this gene in limb development.

Segmental maternal uniparental disomy 7q associated with DLK1/GTL2 (14q32) hypomethylation.

Aberrant methylation at different imprinted loci has been reported for several congenital imprinting disorders, that is, Silver-Russell syndrome (SRS), but the coincidental occurrence of aberrant methylation and uniparental disomy (UPD) has not yet been described. We report on a patient initially diagnosed with SRS carrying a segmental maternal UPD of chromosome 7 [upd(7q)mat]. By further screening the patient's DNA for methylation defects on other chromosomes we identified a hypomethylation of the paternally methylated DLK1/GTL2 locus in 14q32, an epigenotype typically associated with the upd(14)mat phenotype. Detailed clinical analysis confirmed the molecular finding in the patient indicating that the 14q32 epimutation was clinically preponderant. The parallel occurrence of upd(7q)mat and a DLK1/GTL2 hypomethylation in the same patient is a unique finding. Indeed, both disturbances might have occurred coincidentally, but it can also be hypothesized that the upd(7q)mat as the initial genomic mutation represents a trans-acting mutation causing an aberrant methylation in 14q32.

Genetic and epigenetic findings in Silver-Russell syndrome.

Silver-Russell syndrome (SRS) is a genetically and clinically heterogeneous disease which is mainly characterized by pre- and postnatal growth restriction. The typical SRS phenotype furthermore includes a relative macrocephaly, a triangular shaped face, body asymmetry, clinodactyly of the fifth finger and other less constant features. In about ~50% of patients (epi)genetic alterations involving chromosomes 7 and 11 can be detected. The major finding (~44%) is a hypomethylation of the imprinting control region 1 (ICR1) in 11p15.5 affecting the expression of H19 and IGF2. 4-10% of the patients carry a maternal UPD of chromosome 7 (UPD(7)mat). In a few cases chromosomal rearrangements have been reported. The diagnostic workup should therefore include 11p15 testing, UPD(7)mat analysis and molecular karyotyping. The recurrence risk is generally low in SRS but it can strongly increase in case of familial epimutations or chromosomal rearrangements. Interestingly, in ~7% of 11p15 hypomethylation carriers, hypomethylation of additional imprinted loci can be detected. Clinically, patients with hypomethylation at multiple loci do not differ from those with isolated ICR1 hypomethylation whereas the UPD(7)mat patients generally show a milder phenotype. Nevertheless, an unambiguous (epi)genotype-phenotype correlation can not be delineated. Furthermore, the pathophysiological mechanisms resulting in the SRS phenotype still remain unknown despite the recent progress in deciphering molecular defects in the disease.

New insights into the imprinted MEG8-DMR in 14q32 and clinical and molecular description of novel patients with Temple syndrome.

The chromosomal region 14q32 contains several imprinted genes, which are expressed either from the paternal (DLK1 and RTL1) or the maternal (MEG3, RTL1as and MEG8) allele only. Imprinted expression of these genes is regulated by two differentially methylated regions (DMRs), the germline DLK1/MEG3 intergenic (IG)-DMR (MEG3/DLK1:IG-DMR) and the somatic MEG3-DMR (MEG3:TSS-DMR), which are methylated on the paternal and unmethylated on the maternal allele. Disruption of imprinting in the 14q32 region results in two clinically distinct imprinting disorders, Temple syndrome (TS14) and Kagami-Ogata syndrome (KOS14). Another DMR with a yet unknown function is located in intron 2 of MEG8 (MEG8-DMR, MEG8:Int2-DMR). In contrast to the IG-DMR and the MEG3-DMR, this somatic DMR is methylated on the maternal chromosome and unmethylated on the paternal chromosome. We have performed extensive methylation analyses by deep bisulfite sequencing of the IG-DMR, MEG3-DMR and MEG8-DMR in different prenatal tissues including amniotic fluid cells and chorionic villi. In addition, we have studied the methylation pattern of the MEG8-DMR in different postnatal tissues. We show that the MEG8-DMR is hypermethylated in each of 13 non-deletion TS14 patients (seven newly identified and six previously published patients), irrespective of the underlying molecular cause, and is always hypomethylated in the four patients with KOS14, who have different deletions not encompassing the MEG8-DMR itself. The size and the extent of the deletions and the resulting methylation pattern suggest that transcription starting from the MEG3 promoter may be necessary to establish the methylation imprint at the MEG8-DMR.

Two cases with severe lethal course of Costello syndrome associated with HRAS p.G12C and p.G12D.

Costello syndrome (CS) is a rare congenital disorder characterized by severe failure to thrive, coarse facial appearance, cardiac and skin abnormalities, developmental delay, intellectual disability, and predisposition to malignancies. Heterozygous de novo germline mutations in the proto-oncogene HRAS cause CS. About 80% of patients share the same mutation resulting in the amino acid change p.G12S and present a relatively homogeneous phenotype. Other less common lesions in HRAS can induce a milder phenotype on the one hand and a more severe phenotype on the other broadening the spectrum of clinical manifestations in CS-affected individuals. We report two new patients with the HRAS p.G12C and p.G12D substitutions and a severe neonatal manifestation causing death at the age of three months and 13 days, respectively. Both patients had particularly severe heart involvement with hypertrophic cardiomyopathy and tachyarrhythmia, generalized edema, and respiratory distress. In one case, hypertrophic cardiomyopathy was already noted prenatally. These cases together with other individuals harboring the rare HRAS mutations p.G12C, p.G12V, p.G12D, and p.G12E provide further evidence for a genotype-phenotype correlation that could be of importance for counseling and medical management.