Clinical spectrum of SIX3-associated mutations in holoprosencephaly: correlation between genotype, phenotype and function.

BACKGROUND: Holoprosencephaly (HPE) is the most common structural malformation of the human forebrain. There are several important HPE mutational target genes, including the transcription factor SIX3, which encodes an early regulator of Shh, Wnt, Bmp and Nodal signalling expressed in the developing forebrain and eyes of all vertebrates. OBJECTIVE: To characterise genetic and clinical findings in patients with SIX3 mutations. METHODS: Patients with HPE and their family members were tested for mutations in HPE-associated genes and the genetic and clinical findings, including those for additional cases found in the literature, were analysed. The results were correlated with a mutation-specific functional assay in zebrafish. RESULTS: In a cohort of patients (n = 800) with HPE, SIX3 mutations were found in 4.7% of probands and additional cases were found through testing of relatives. In total, 138 cases of HPE were identified, 59 of whom had not previously been clinically presented. Mutations in SIX3 result in more severe HPE than in other cases of non-chromosomal, non-syndromic HPE. An over-representation of severe HPE was found in patients whose mutations confer greater loss of function, as measured by the functional zebrafish assay. The gender ratio in this combined set of patients was 1.5:1 (F:M) and maternal inheritance was almost twice as common as paternal. About 14% of SIX3 mutations in probands occur de novo. There is a wide intrafamilial clinical range of features and classical penetrance is estimated to be at least 62%. CONCLUSIONS: Our data suggest that SIX3 mutations result in relatively severe HPE and that there is a genotype-phenotype correlation, as shown by functional studies using animal models.

Microdeletion 15q13.3: a locus with incomplete penetrance for autism, mental retardation, and psychiatric disorders.

BACKGROUND: Microdeletions within chromosome 15q13.3 are associated both with a recently recognised syndrome of mental retardation, seizures, and dysmorphic features, and with schizophrenia. METHODS AND RESULTS: Based on routine diagnostic testing of approximately 8200 samples using array comparative genomic hybridisation, we identified 20 individuals (14 children and six parents in 12 families) with microdeletions of 15q13.3. Phenotypes in the children included developmental delay, mental retardation, or borderline IQ in most and autistic spectrum disorder (6/14), speech delay, aggressiveness, attention deficit hyperactivity disorder, and other behavioural problems. Both parents were available in seven families, and the deletion was de novo in one, inherited from an apparently normal parent in four, and inherited from a parent with learning disability and bipolar disorder in two families. Of the 14 children, six in five families were adopted, and DNA was available for only one of these 10 biological parents; the deletion was very likely inherited for one of these families with two affected children. Among the unavailable parents, two mothers were described as having mental retardation, another mother as having "mental illness", and one father as having schizophrenia. We hypothesise that some of the unavailable parents have the deletion. CONCLUSIONS: The occurrence of increased adoption, frequent autism, bipolar disorder, and lack of penetrance are noteworthy findings in individuals with deletion 15q13.3. A high rate of adoption may be related to the presence of the deletion in biological parents. Unconfirmed histories of antisocial behaviours in unavailable biological parents raise the concern that future research may show that deletion 15q13.3 is associated with such behaviours.

20p12.3 microdeletion predisposes to Wolff-Parkinson-White syndrome with variable neurocognitive deficits.

BACKGROUND: Wolff-Parkinson-White syndrome (WPW) is a bypass re-entrant tachycardia that results from an abnormal connection between the atria and ventricles. Mutations in PRKAG2 have been described in patients with familial WPW syndrome and hypertrophic cardiomyopathy. Based on the role of bone morphogenetic protein (BMP) signalling in the development of annulus fibrosus in mice, it has been proposed that BMP signalling through the type 1a receptor and other downstream components may play a role in pre-excitation. METHODS AND RESULTS: Using the array comparative genomic hybridisation (CGH), we identified five individuals with non-recurrent deletions of 20p12.3. Four of these individuals had WPW syndrome with variable dysmorphisms and neurocognitive delay. With the exception of one maternally inherited deletion, all occurred de novo, and the smallest of these harboured a single gene, BMP2. In two individuals with additional features of Alagille syndrome, deletion of both JAG1 and BMP2 were identified. Deletion of this region has not been described as a copy number variant in the Database of Genomic Variants and has not been identified in 13 321 individuals from other cohort examined by array CGH in our laboratory. CONCLUSIONS: Our findings demonstrate a novel genomic disorder characterised by deletion of BMP2 with variable cognitive deficits and dysmorphic features and show that individuals bearing microdeletions in 20p12.3 often present with WPW syndrome.

Congenital diaphragmatic hernia in WAGR syndrome.

Wilms tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR) syndrome is a contiguous gene deletion syndrome involving the Wilms tumor 1 gene (WT1), the paired box gene 6 (PAX6), and possibly other genes on chromosome 11p13. WT1 is required for normal formation of the genitourinary system and the high incidence of Wilms tumor and genitourinary anomalies found in patients with WAGR are attributed to haploinsufficiency of this gene. It has been hypothesized that WT1 also plays an important role in the development of the diaphragm. During mammalian embryonic development, WT1 is expressed in the pleural and abdominal mesothelium that forms part of the diaphragm. Furthermore, mice that are homozygous for a deletion in the mouse homolog of WT1 have diaphragmatic hernias. Case reports describing congenital diaphragmatic hernias in infants with Denys-Drash and Frasier syndromes, both of which can be caused by mutations in WT1, provide additional support for this hypothesis. We report an infant with aniridia, bilateral cryptorchidism, vesicoureteral reflux, and a right-sided Morgagni-type diaphragmatic hernia. G-banded chromosome analysis revealed a deletion of 11p12-p15.1. Breakpoint regions were refined by fluorescence in situ hybridization (FISH) and deletion of the WAGR critical region, including WT1, was confirmed. A review of the medical literature identified a second patient with a deletion of 11p13, a left-sided Bochdalek-type diaphragmatic hernia, and anomalies that suggest a diagnosis of WAGR including bilateral microphthalmia, a small penis, bilateral cryptorchidism, and a hypoplastic scrotum. These cases demonstrate that congenital diaphragmatic hernia can be associated with WAGR syndrome and suggest that deletions of WT1 may predispose individuals to develop congenital diaphragmatic hernia.

Mutations in the neurofilament light chain gene (NEFL) cause early onset severe Charcot-Marie-Tooth disease.

Neurofilament light chain polypeptide (NEFL) is one of the most abundant cytoskeletal components of the neuron. Mutations in the NEFL gene were recently reported as a cause for autosomal dominant Charcot-Marie-Tooth type 2E (CMT2E) linked to chromosome 8p21. In order to investigate the frequency and phenotypic consequences of NEFL mutations, we screened 323 patients with CMT or related peripheral neuropathies. We detected six disease associated missense mutations and one 3-bp in-frame deletion clustered in functionally defined domains of the NEFL protein. Patients have an early onset and often a severe clinical phenotype. Electrophysiological examination shows moderately to severely slowed nerve conduction velocities. We report the first nerve biopsy of a CMT patient with a de novo missense mutation in NEFL, and found an axonal pathology with axonal regeneration clusters and onion bulb formations. Our findings provide further evidence that the clinical variation observed in CMT depends on the gene mutated and the specific type of mutation, and we also suggest that NEFL mutations need to be considered in the molecular evaluation of patients with sporadic or dominantly inherited CMT.

Novel mutations of MYO15A associated with profound deafness in consanguineous families and moderately severe hearing loss in a patient with Smith-Magenis syndrome.

Mutations in myosin XVA are responsible for the shaker 2 ( sh2) phenotype in mice and nonsyndromic autosomal recessive profound hearing loss DFNB3 on chromosome 17p11.2. We have ascertained seven families with profound congenital hearing loss from Pakistan and India with evidence of linkage to DFNB3 at 17p11.2. We report three novel homozygous mutations in MYO15A segregating in three of these families. In addition, one hemizygous missense mutation of MYO15A was found in one of eight Smith-Magenis syndrome (del(17)p11.2) patients from North America who had moderately severe sensorineural hearing loss.

A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster.

We performed a systematic analysis of 929 human disease gene entries associated with at least one mutant allele in the Online Mendelian Inheritance in Man (OMIM) database against the recently completed genome sequence of Drosophila melanogaster. The results of this search have been formatted as an updateable and searchable on-line database called Homophila. Our analysis identified 714 distinct human disease genes (77% of disease genes searched) matching 548 unique Drosophila sequences, which we have summarized by disease category. This breakdown into disease classes creates a picture of disease genes that are amenable to study using Drosophila as the model organism. Of the 548 Drosophila genes related to human disease genes, 153 are associated with known mutant alleles and 56 more are tagged by P-element insertions in or near the gene. Examples of how to use the database to identify Drosophila genes related to human disease genes are presented. We anticipate that cross-genomic analysis of human disease genes using the power of Drosophila second-site modifier screens will promote interaction between human and Drosophila research groups, accelerating the understanding of the pathogenesis of human genetic disease. The Homophila database is available at http://homophila.sdsc.edu.

Trisomy 17p10-p12 resulting from a supernumerary marker chromosome derived from chromosome 17: molecular analysis and delineation of the phenotype.

We report a 5-year-old boy with a small de novo marker chromosome derived from the proximal short arm of chromosome 17. His clinical features include hypotonia, global developmental delay, oval face with large nose and prominent ears, and ligamentous laxity of the fingers. Magnetic resonance imaging of the brain demonstrated mildly delayed myelination. G-band chromosome analysis revealed mosaicism for a small marker chromosome in 85% of the peripheral blood cells analyzed. Fluorescence in situ hybridization and microsatellite polymorphism studies showed that the der(17) was of maternal origin and included genetic material from the 17p10-p12 region, but did not contain the PMP22 gene. One breakpoint mapped within the centromere and the second breakpoint mapped adjacent to the Charcot-Marie-Tooth disease type 1A proximal low-copy repeat (CMT1A-REP). We compare the clinical characteristics of our patient with those previously reported to have a duplication involving the proximal short arm region of chromosome 17 to further delineate the phenotype of trisomy 17pl0-p12.

Haploinsufficiency of ALX4 as a potential cause of parietal foramina in the 11p11.2 contiguous gene-deletion syndrome.

Heterozygous mutations in MSX2 are responsible for an autosomal dominant form of parietal foramina (PFM). PFM are oval defects of the parietal bones that are also a characteristic feature of a contiguous gene-deletion syndrome caused by a proximal deletion in the short arm of chromosome 11 (Potocki-Shaffer syndrome). We have identified a human bacterial artificial chromosome (BAC) clone mapping to chromosome 11, containing a region homologous to the human homeobox gene MSX2. Further sequence analysis demonstrated that the human orthologue (ALX4) of the mouse Aristaless-like 4 gene (Alx4) is contained within this 11p clone. We used FISH to test for the presence-or for the heterozygous deletion-of this clone in two patients with the 11p11.2-deletion syndrome and showed that this clone is deleted in these patients. ALX4 and Alx4 were shown to be expressed in bone and to be absent from all other tissues tested. The involvement of Alx4 in murine skull development, its bone-specific expression pattern, the fact that Alx4 is a dosage-sensitive gene in mice, and the localization of a human genomic clone containing ALX4 to 11p11.2, with hemizygosity in patients with deletion of 11p11.2 who have biparietal foramina, support the contention that ALX4 is a candidate gene for the PFM in the 11p11.2-deletion syndrome.

Circadian rhythm abnormalities of melatonin in Smith-Magenis syndrome.

BACKGROUND: Smith-Magenis syndrome (SMS) is a multiple congenital anomalies/mental retardation syndrome associated with a hemizygous deletion of chromosome 17, band p11.2. Characteristic features include neurobehavioural abnormalities such as aggressive and self-injurious behaviour and significant sleep disturbances. The majority of patients have a common deletion characterised at the molecular level. Physical mapping studies indicate that all patients with the common deletion are haploinsufficient for subunit 3 of the COP9 signalosome (COPS3), which is conserved from plants to humans, and in the plant Arabidopis thaliana regulates gene transcription in response to light. Haploinsufficiency of this gene is hypothesised to be potentially involved in the sleep disturbances seen in these patients. Melatonin is a hormone secreted by the pineal gland. SMS patients are reported to have fewer sleep disturbances when given a night time dose of this sleep inducing hormone. METHODS: Urinary excretion of 6-sulphatoxymelatonin (aMT6s), the major hepatic metabolite of melatonin, in 19 SMS patients were measured in conjunction with 24 hour sleep studies in 28 SMS patients. Five of the 28 patients did not have the common SMS deletion. To investigate a potential correlation of COPS3 haploinsufficiency and disturbed melatonin excretion, we performed fluorescence in situ hybridisation (FISH) using two BACs containing coding exons of COPS3. RESULTS: All SMS patients show significant sleep disturbances when assessed by objective criteria. Abnormalities in the circadian rhythm of aMT6s were observed in all but one SMS patient. Interestingly this patient did not have the common deletion. All patients studied, including the one patient with a normal melatonin rhythm, were haploinsufficient for COPS3. CONCLUSIONS: Our data indicate a disturbed circadian rhythm in melatonin and document the disturbed sleep pattern in Smith-Magenis syndrome. Our findings suggest that the abnormalities in the circadian rhythm of melatonin and altered sleep patterns could be secondary to aberrations in the production, secretion, distribution, or metabolism of melatonin; however, a direct role for COPS3 could not be established.

Molecular mechanism for duplication 17p11.2- the homologous recombination reciprocal of the Smith-Magenis microdeletion.

Recombination between repeated sequences at various loci of the human genome are known to give rise to DNA rearrangements associated with many genetic disorders. Perhaps the most extensively characterized genomic region prone to rearrangement is 17p12, which is associated with the peripheral neuropathies, hereditary neuropathy with liability to pressure palsies (HNPP) and Charcot-Marie-Tooth disease type 1A (CMT1A;ref. 2). Homologous recombination between 24-kb flanking repeats, termed CMT1A-REPs, results in a 1.5-Mb deletion that is associated with HNPP, and the reciprocal duplication product is associated with CMT1A (ref. 2). Smith-Magenis syndrome (SMS) is a multiple congenital anomalies, mental retardation syndrome associated with a chromosome 17 microdeletion, del(17)(p11.2p11.2) (ref. 3,4). Most patients (>90%) carry deletions of the same genetic markers and define a common deletion. We report seven unrelated patients with de novo duplications of the same region deleted in SMS. A unique junction fragment, of the same apparent size, was identified in each patient by pulsed field gel electrophoresis (PFGE). Further molecular analyses suggest that the de novo17p11.2 duplication is preferentially paternal in origin, arises from unequal crossing over due to homologous recombination between flanking repeat gene clusters and probably represents the reciprocal recombination product of the SMS deletion. The clinical phenotype resulting from duplication [dup(17)(p11.2p11.2)] is milder than that associated with deficiency of this genomic region. This mechanism of reciprocal deletion and duplication via homologous recombination may not only pertain to the 17p11.2 region, but may also be common to other regions of the genome where interstitial microdeletion syndromes have been defined.

DNA rearrangements on both homologues of chromosome 17 in a mildly delayed individual with a family history of autosomal dominant carpal tunnel syndrome.

Disorders known to be caused by molecular and cytogenetic abnormalities of the proximal short arm of chromosome 17 include Charcot-Marie-Tooth disease type 1A (CMT1A), hereditary neuropathy with liability to pressure palsies (HNPP), Smith-Magenis syndrome (SMS), and mental retardation and congenital anomalies associated with partial duplication of 17p. We identified a patient with multifocal mononeuropathies and mild distal neuropathy, growth hormone deficiency, and mild mental retardation who was found to have a duplication of the SMS region of 17p11.2 and a deletion of the peripheral myelin protein 22 (PMP22) gene within 17p12 on the homologous chromosome. Further molecular analyses reveal that the dup(17)(p11.2p11.2) is a de novo event but that the PMP22 deletion is familial. The family members with deletions of PMP22 have abnormalities indicative of carpal tunnel syndrome, documented by electrophysiological studies prior to molecular analysis. The chromosomal duplication was shown by interphase FISH analysis to be a tandem duplication. These data indicate that familial entrapment neuropathies, such as carpal tunnel syndrome and focal ulnar neuropathy syndrome, can occur because of deletions of the PMP22 gene. The co-occurrence of the 17p11.2 duplication and the PMP22 deletion in this patient likely reflects the relatively high frequency at which these abnormalities arise and the underlying molecular characteristics of the genome in this region.

Delineation of the common critical region in Williams syndrome and clinical correlation of growth, heart defects, ethnicity, and parental origin.

Williams syndrome (WS) is a neurodevelopmental disorder with a variable phenotype. Molecular genetic studies have indicated that hemizygosity at the elastin locus (ELN) may account for the cardiac abnormalities seen in WS, but that mental retardation and hypercalcemia are likely caused by other genes flanking ELN. In this study, we defined the minimal critical deletion region in 63 patients using 10 microsatellite markers and 5 fluorescence in situ hybridization (FISH) probes on chromosome 7q, flanking ELN. The haplotype analyses showed the deleted cases to have deletions of consistent size, as did the FISH analyses using genomic probes for the known ends of the commonly deleted region defined by the satellite markers. In all informative cases deleted at ELN, the deletion extends from D7S489U to D7S1870. The genetic distance between these two markers is about 2 cM. Of the 51 informative patients with deletions, 29 were maternal and 22 were paternal in origin. There was no evidence for effects on stature by examining gender, ethnicity, cardiac status, or parental origin of the deletion. Heteroduplex analysis for LIMK1, a candidate gene previously implicated in the WS phenotype, did not show any mutations in our WS patients not deleted for ELN. LIMK1 deletions were found in all elastin-deletion cases who had WS. One case, who has isolated, supravalvular aortic stenosis and an elastin deletion, was not deleted for LIMK1. It remains to be determined if haploinsufficiency of LIMK1 is responsible in part for the WS phenotype or is simply deleted due to its close proximity to the elastin locus.

Homologous recombination of a flanking repeat gene cluster is a mechanism for a common contiguous gene deletion syndrome.

Smith-Magenis syndrome (SMS), caused by del(17)p11.2, represents one of the most frequently observed human microdeletion syndromes. We have identified three copies of a low-copy-number repeat (SMS-REPs) located within and flanking the SMS common deletion region and show that SMS-REP represents a repeated gene cluster. We have isolated a corresponding cDNA clone that identifies a novel junction fragment from 29 unrelated SMS patients and a different-sized junction fragment from a patient with dup(17)p11.2. Our results suggest that homologous recombination of a flanking repeat gene cluster is a mechanism for this common microdeletion syndrome.

Quantification by flow cytometry of chromosome-17 deletions in Smith-Magenis syndrome patients.

We have used bivariate flow karyotyping to quantify the deletions involving chromosome 17 in sixteen patients with Smith-Magenis syndrome (SMS). The fluorescence intensities of mitotic chromosomes stained with Hoechst 33258 and chromomycin were quantified in a dual-beam flow cytometer. For each patient, the position of the peak representing the deleted chromosome 17 was compared to those of the normal homologs of an unaffected parent. The patients could be classified into four groups based on the size of their deletions. The deletions ranged from approximately 9-10 Mb (approximately 10-11% of the chromosome) to below the detection limit of the technique (2 Mb). Different deletion sizes were detected among patients whose high-resolution banding results were similar. Some deletions detected by banding were not detected by flow analyses. Deletion estimates are largely consistent with the results of molecular analyses. Patients with larger deletions that extend into band 17p 12 have abnormal electrophysiologic studies of peripheral nerves. Deletion size does not appear to correlate with the degree of mental retardation, presence of behavioral abnormalities, craniofacial anomalies or common skeletal findings in SMS. By identifying patients with varying deletion sizes, these data will aid the construction of a long-range deletion-based map of 17p11.2 and identification of the genes involved in this syndrome.

Multi-disciplinary clinical study of Smith-Magenis syndrome (deletion 17p11.2)

Smith-Magenis syndrome (SMS) is a multiple congenital anomaly, mental retardation (MCA/MR) syndrome associated with deletion of chromosome 17 band p11.2. As part of a multi-disciplinary clinical, cytogenetic, and molecular approach to SMS, detailed clinical studies including radiographic, neurologic, developmental, ophthalmologic, otolaryngologic, and audiologic evaluations were performed on 27 SMS patients. Significant findings include otolaryngologic abnormalities in 94%, eye abnormalities in 85%, sleep abnormalities (especially reduced REM sleep) in 75%, hearing impairment in 68% (approximately 65% conductive and 35% sensorineural), scoliosis in 65%, brain abnormalities (predominantly ventriculomegaly) in 52%, cardiac abnormalities in at least 37%, renal anomalies (especially duplication of the collecting system) in 35%, low thyroxine levels in 29%, low immunoglobulin levels in 23%, and forearm abnormalities in 16%. The measured IQ ranged between 20-78, most patients falling in the moderate range of mental retardation at 40-54, although several patients scored in the mild or borderline range. The frequency of these many abnormalities in SMS suggests that patients should be evaluated thoroughly for associated complications both at the time of diagnosis and at least annually thereafter.

Interstitial deletion of 11(p11.2p12): a newly described contiguous gene deletion syndrome involving the gene for hereditary multiple exostoses (EXT2).

Individuals with deletions of the proximal portion of the short arm of chromosome 11 share many manifestations including mental retardation, biparietal foramina, minor facial anomalies, and multiple cartilaginous exostoses. The finding of multiple exostoses in these patients is remarkable as the disorder hereditary multiple exostoses, which is inherited in an autosomal dominant manner, has recently been mapped by linkage to three regions, including proximal 11p. We report the clinical and molecular findings in an additional patient with an 11(p11.2p12) deletion. Cytogenetic and molecular analysis demonstrated a de novo, paternally derived deletion for markers which have been shown to be tightly linked to the 11p locus (EXT2). These data support the location of EXT2 within this region and also provide information regarding the ordering of polymorphic markers on 11p. Deletion 11(p11.2p12) is a rare, yet specific, deletion syndrome involving the EXT2 locus, a gene for parietal foramina, and a mental retardation locus, and therefore can be classified as a contiguous gene deletion syndrome.