Delayed diagnosis of adrenal hypoplasia congenita in a patient with a new mutation in the NR0B1 gene.

Determining the precise cause of adrenal insufficiency occurring in infancy is of critical importance for both the correct management of affected children and the provision of correct genetic advice to their families. We report a case of a 24-year-old, male patient bearing a new mutation in the DAX1 gene. The patient was born at term, from a healthy pregnancy. Adrenal insufficiency was diagnosed in the fourth week of life with a salt-wasting syndrome, but it was mistakenly believed to be secondary to congenital adrenal hyperplasia (CAH). On hydrocortisone substitution, the child continued to develop normally, but the diagnosis of CAH was questioned, which led to an episode of an abrupt withdrawal of hydrocortisone substitution and subsequently caused a reoccurrence of a life-threatening salt-wasting syndrome. Owing to close follow-up, the patient's gonadal axis deficiency was promptly identified, which allowed an assisted but successful onset of puberty. We proposed the diagnosis of adrenal hypoplasia congenita (AHC) in this patient and identified a hemizygous mutation (c.1130delAinsGT, p.E377GfsX12) in exon 1 of the NR0B1 gene. To our knowledge, the detected mutation has not been described previously (HGMD Professional 2010.4, Human Gene Mutation Database, Biobase, Beverly, MA, USA). It leads to a frameshift, a premature stop codon, and, most likely, non-sense-mediated decay of the mutant mRNA. In this case, close patient follow-up minimized the detrimental consequences of an incorrect diagnosis. Nevertheless, it highlights the importance of the early precise diagnosis of patients with AHC.

Expanding the phenotypic spectrum of lupus erythematosus in Aicardi-Goutières syndrome.

OBJECTIVE: Aicardi-Goutières syndrome (AGS) is an early-onset encephalopathy resembling congenital viral infection that is characterized by basal ganglia calcifications, loss of white matter, cerebrospinal fluid (CSF) lymphocytosis, and elevated interferon-alpha levels in the CSF. Studies have shown that AGS is an autosomal-recessive disease linked to mutations in 5 genes, encoding the 3'-repair DNA exonuclease 1 (TREX1), the 3 subunits of ribonuclease H2 (RNASEH2A-C), and sterile alpha motif domain and HD domain-containing protein 1 (SAMHD1). In this study we further characterized the phenotypic spectrum of this disease. METHODS: Clinical and laboratory data were obtained from 26 patients fulfilling the clinical diagnostic criteria for AGS. Genomic DNA was screened for mutations in all 5 AGS genes by direct sequencing, and sera were analyzed for autoantibodies. RESULTS: In 20 patients with AGS, 20 mutations, 12 of which were novel, were identified in all 5 AGS genes. Clinical and laboratory investigations revealed a high prevalence of features (some not previously described in patients with AGS) that are commonly seen in patients with systemic lupus erythematosus (SLE), such as thrombocytopenia, leukocytopenia, antinuclear antibodies, erythematous lesions, oral ulcers, and arthritis, which were observed in 12 (60%) of 20 patients with AGS. Moreover, the coexistence of AGS and SLE, was for the first time, demonstrated in 2 patients with molecularly proven AGS. CONCLUSION: These findings expand the phenotypic spectrum of lupus erythematosus in AGS and provide further insight into its disease mechanisms by showing that activation of the innate immune system as a result of inherited defects in nucleic acid metabolism could lead to systemic autoimmunity.

Sall1, sall2, and sall4 are required for neural tube closure in mice.

Four homologs to the Drosophila homeotic gene spalt (sal) exist in both humans and mice (SALL1 to SALL4/Sall1 to Sall4, respectively). Mutations in both SALL1 and SALL4 result in the autosomal-dominant developmental disorders Townes-Brocks and Okihiro syndrome, respectively. In contrast, no human diseases have been associated with SALL2 to date, and Sall2-deficient mice have shown no apparent abnormal phenotype. We generated mice deficient in Sall2 and, contrary to previous reports, 11% of our Sall2-deficient mice showed background-specific neural tube defects, suggesting that Sall2 has a role in neurogenesis. To investigate whether Sall4 may compensate for the absence of Sall2, we generated compound Sall2 knockout/Sall4 genetrap mutant mice. In these mutants, the incidence of neural tube defects was significantly increased. Furthermore, we found a similar phenotype in compound Sall1/4 mutant mice, and in vitro studies showed that SALL1, SALL2, and SALL4 all co-localized in the nucleus. We therefore suggest a fundamental and redundant function of the Sall proteins in murine neurulation, with the heterozygous loss of a particular SALL protein also possibly compensated in humans during development.

Tetra-amelia and lung hypo/aplasia syndrome: new case report and review.

Tetra-amelia is a rare malformation that may be associated with other anomalies and is usually inherited in an autosomal recessive pattern. We describe a fetus, born to a nonconsanguineous couple, with tetra-amelia, bilateral cleft lip and palate and bilateral lung agenesis, without other anomalies. Karyotype was normal (46,XX) and premature centromere separation was excluded. No mutation was identified upon molecular analysis of WNT3, HS6ST1, and HS6ST3. We reviewed the literature and the differential diagnosis to clarify the clinical delineation of conditions associated with tetra-amelia. The present report describes the sixth family with this pattern of malformations and reinforces the evidence that the "tetra-amelia and lung hypo/aplasia syndrome" is a distinct autosomal recessive condition, with no identified gene thus far.

Multigene deletions on chromosome 20q13.13-q13.2 including SALL4 result in an expanded phenotype of Okihiro syndrome plus developmental delay.

Okihiro syndrome results from truncating mutations in the SALL4 locus on the chromosome 20q13.13-q13.2. Deletions of the whole SALL4 coding region as well as single exon deletions are also a common cause of Okihiro syndrome and indicate haploinsufficiency as the disease causing mechanism. The phenotypes caused by SALL4 deletions are not different from those caused by point mutations. No multigene deletion including SALL4 has been documented to date. Here we report the detection and molecular characterization of four novel, overlapping microdeletions, all spanning SALL4 and flanking genes, in four unrelated cases with features of Okihiro syndrome and variable degrees of psychomotor delay. All deletions were first identified and mapped by quantitative Real Time PCR. Subsequently, three of four deletions were mapped in further detail by high-resolution array CGH (244k oligo-arrays). All cases had larger deletions of varying size (1.76-1.78 Mb, 2.01-2.05 Mb, 2.16-2.17 Mb, and 1.3-2.8 Mb, respectively), which included SALL4 plus 3 to 7 additional functional genes. While three cases with largely overlapping deletions are mildly developmentally delayed, the only patient with a more centromeric deletion is clearly mentally retarded. In this patient, four genes (MOCS3, DPM1, ADNP, BCAS4) are deleted, which were not affected in the other three cases, suggesting that the deletion of one or more of these genes contributes to the mental retardation. Since two of the four cases presented with choanal atresia, large deletions including SALL4 should be considered in the differential diagnosis of children with suspected CHARGE syndrome but without detectable CHD7 mutations.

Germline NF1 mutational spectra and loss-of-heterozygosity analyses in patients with pheochromocytoma and neurofibromatosis type 1.

BACKGROUND: Neurofibromatosis type 1 (NF1) is a pheochromocytoma-associated syndrome. Because of the low prevalence of pheochromocytoma in NF1, we ascertained subjects by pheochromocytoma that also had NF1 in the hope of describing the germline NF1 mutational spectra of NF1-related pheochromocytoma. MATERIALS AND METHODS: An international registry for NF1-pheochromocytomas was established. Mutation scanning was performed using denaturing HPLC for intragenic variation and quantitative PCR for large deletions. Loss-of-heterozygosity analysis using markers in and around NF1 was performed. RESULTS: There were 37 eligible subjects (ages 14-70 yr). Of 21 patients with corresponding tumor available, 67% showed somatic loss of the nonmutated allele at the NF1 locus vs. 0 of 12 sporadic tumors (P = 0.0002). Overall, 86% of the 37 patients had exonic or splice site mutations, 14% large deletions or duplications; 79% of the mutations are novel. The cysteine-serine rich domain (CSR) was affected in 35% but the RAS GTPase activating protein domain (RGD) in only 13%. There did not appear to be an association between any clinical features, particularly pheochromocytoma presentation and severity, and NF1 mutation genotype. CONCLUSIONS: The germline NF1 mutational spectra comprise intragenic mutations and deletions in individuals with pheochromocytoma and NF1. NF1 mutations tended to cluster in the CSR over the RAS-GAP domain, suggesting that CSR plays a more prominent role in individuals with NF1-pheochromocytoma than in NF1 individuals without this tumor. Loss-of-heterozygosity of NF1 markers in NF1-related pheochromocytoma was significantly more frequent than in sporadic pheochromocytoma, providing further molecular evidence that pheochromocytoma is a true component of NF1.

Molecular analysis of the CHD7 gene in CHARGE syndrome: identification of 22 novel mutations and evidence for a low contribution of large CHD7 deletions.

PURPOSE: Autosomal dominant CHARGE syndrome (OMIM no. 214800) is characterized by choanal atresia or cleft lip or palate, ocular colobomas, cardiovascular malformations, retardation of growth, ear anomalies, and deafness, and is caused by mutations in the CHD7 gene. Here, we describe the outcome of a molecular genetic analysis in 18 Finnish and 56 German patients referred for molecular confirmation of the clinical diagnosis of suspected CHARGE syndrome. METHODS: Quantitative real-time polymerase chain reaction or multiplex ligation-dependent probe amplification assays did not reveal deletions in mutation negative cases, suggesting that larger CHD7 deletions are not a major cause of CHARGE syndrome. RESULTS: In this group of 74 patients, we found mutations in 30 cases. 22 mutations were novel, including 11 frameshift, 5 nonsense, 3 splice-site, and 3 missense mutations. One de novo frameshift mutation was found in the last exon and is expected to result in a minimally shortened CHD7 polypeptide. Because the mutation is associated with a typical CHARGE syndrome phenotype, it may indicate the presence of an as yet unknown functional domain in the very carboxyterminal end of CHD7. CONCLUSIONS: Our mutation detection rate of 40.5% is reflective of screening an unselected sample population referred for CHD7 testing based on suspected clinical diagnosis of CHARGE syndrome and not for having met strict clinical criteria for this disorder.

Detection of heterozygous SALL1 deletions by quantitative real time PCR proves the contribution of a SALL1 dosage effect in the pathogenesis of Townes-Brocks syndrome.

Townes-Brocks syndrome (TBS) is an autosomal dominantly inherited disorder characterized by ear, anal, limb, and renal malformations, and results from mutations in the gene SALL1. All SALL1 mutations previously found in TBS patients create preterminal termination codons. In accordance with the findings of pericentric inversions or balanced translocations, TBS was initially assumed to be caused by SALL1 haploinsufficiency. This assumption was strongly contradicted by a Sall1 mouse knock-out, because neither hetero- nor homozygous knock-out mutants displayed a TBS-like phenotype. A different mouse mutant mimicking the human SALL1 mutations, however, showed a TBS-like phenotype in the heterozygous situation, suggesting a dominant-negative action of the mutations causing TBS. We applied quantitative real time PCR to detect and map SALL1 deletions in 240 patients with the clinical diagnosis of TBS, who were negative for SALL1 mutations. Deletions were found in three families. In the first family, a 75 kb deletion including all SALL1 exons had been inherited by two siblings from their father. A second, sporadic patient carried a de novo 1.9-2.6 Mb deletion including the whole SALL1 gene, and yet another sporadic case was found to carry an intragenic deletion of 3384 bp. In all affected persons, the TBS phenotype is rather mild as compared to the phenotype resulting from point mutations. These results confirm that SALL1 haploinsufficiency is sufficient to cause a mild TBS phenotype but suggest that it is not sufficient to cause the severe, classical form. It therefore seems that there is a different contribution of SALL1 gene function to mouse and human embryonic development.

Expanding the spectrum of TBX5 mutations in Holt-Oram syndrome: detection of two intragenic deletions by quantitative real time PCR, and report of eight novel point mutations.

Mutations in the gene TBX5 cause Holt-Oram syndrome (HOS), an autosomal dominant disorder characterized by anterior (i.e., radial ray) upper limb malformations and congenital heart defects and/or cardiac conduction anomalies. The detection rate for TBX5 mutations in HOS patients has been given as 30-35% in most reports. However, a detection rate of 74% was reported when strict clinical inclusion criteria for HOS were applied prior to TBX5 analysis. Still, in a significant proportion of typical HOS cases no mutation can be found within the TBX5 coding region and flanking intronic sequences. One explanation could be that large but submicroscopic deletions of TBX5 could cause HOS, yet only one such TBX5 deletion has been reported to date. We developed a quantitative Real Time PCR strategy to detect large, submicroscopic deletions in TBX5. Using this assay, we screened a total of 102 TBX5 mutation negative patients and discovered two novel intragenic deletions. One deletion of 7756 bp removes exon 6 and a considerable part of the neighboring intronic sequences, and the other of 3695 bp removes exon 9 with the stop codon and the 3'UTR completely as well as a part of the preceding intron 8. We conclude that quantitative Real Time PCR is a reliable method to detect submicroscopic deletions within TBX5. However, such deletions explain only approximately 2% of the TBX5 mutational spectrum in HOS cases. In addition, we also present eight novel TBX5 mutations (three nonsense, one splice mutation, four short deletions) as detected by direct sequencing in 21 families not previously analyzed for mutations.

Frequency of progranulin mutations in a German cohort of 79 frontotemporal dementia patients.

Mutations of the progranulin gene lead to progranulin haploinsufficiency and to frontotemporal lobar degeneration (FTD) with TDP-43 positive inclusions. It is assumed that unknown genetic, epigenetic and environmental factors are responsible for the observed marked degree of phenotypic variability among mutation carriers. This is the first published series of German FTD cases screened for progranulin mutations. Mean age at onset was 62 years, 19 patients (24%) had a positive family history of dementia, and 11 patients (14%) had a positive family history for probable FTD. Data on FTD subtypes are presented. Two mutations were identified (3%), one of which has been described previously. Clinically, both patients showed the frontal-behavioural variant type of FTD. Remarkably, a sibling of one case presented with progressive nonfluent aphasia, clinically distinct from the brother. We also performed quantitative PCR analyses to detect potential whole progranulin gene and exon deletions. Here, results were negative.

Mutations in the cyclin family member FAM58A cause an X-linked dominant disorder characterized by syndactyly, telecanthus and anogenital and renal malformations.

We identified four girls with a consistent constellation of facial dysmorphism and malformations previously reported in a single mother-daughter pair. Toe syndactyly, telecanthus and anogenital and renal malformations were present in all affected individuals; thus, we propose the name 'STAR syndrome' for this disorder. Using array CGH, qPCR and sequence analysis, we found causative mutations in FAM58A on Xq28 in all affected individuals, suggesting an X-linked dominant inheritance pattern for this recognizable syndrome.

Synergistic cooperation of Sall4 and Cyclin D1 in transcriptional repression.

Loss of function mutations in SALL4 cause Okihiro syndrome, an autosomal dominant disorder characterised by radial ray malformations associated with Duane anomaly. In zebrafish and mouse Sall4 interacts with TBX5 during limb and heart development and plays a crucial role for embryonic stem (ES) cell pluripotency. Here we report the nuclear interaction of murine Sall4 with Cyclin D1, one of the main regulators of G(1) to S phase transition in cell cycle, verified by yeast two-hybrid assay, co-immunoprecipitation and intracellular co-localisation. Furthermore, using luciferase reporter gene assays we demonstrate that Sall4 operates as a transcriptional repressor located to heterochromatin and that this activity is modulated by Cyclin D1.

A SALL4 zinc finger missense mutation predicted to result in increased DNA binding affinity is associated with cranial midline defects and mild features of Okihiro syndrome.

Truncating mutations of the gene SALL4 on chromosome 20q13.13-13.2 cause Okihiro and acro-renal-ocular syndromes. Pathogenic missense mutations within the SALL4 or SALL1 genes have not yet been reported, raising the question which phenotypic features would be associated with them. Here we describe the first missense mutation within the SALL4 gene. The mutation results in an exchange of a highly conserved zinc-coordinating Histidine crucial for zinc finger (ZF) structure within a C2H2 double ZF domain to an Arginine. Molecular modeling predicts that this exchange does not result in a loss of zinc ion binding but leads to an increased DNA-binding affinity of the domain. The index patient shows mild features of Okihiro syndrome, but in addition cranial midline defects (pituitary hypoplasia and single central incisor). This finding illustrates that the phenotypic and functional effects of SALL4 missense mutations are difficult to predict, and that other SALL4 missense mutations might lead to phenotypes not overlapping with Okihiro syndrome.