Mutation analysis of the MKKS gene in McKusick-Kaufman syndrome and selected Bardet-Biedl syndrome patients.

McKusick-Kaufman syndrome comprises hydrometrocolpos, polydactyly, and congenital heart defects and overlaps with Bardet-Biedl syndrome, comprising retinitis pigmentosa, polydactyly, obesity, mental retardation, and renal and genital anomalies. Bardet-Biedl syndrome is genetically heterogeneous with three cloned genes ( BBS2, BBS4, and MKKS) and at least three other known loci ( BBS1, BBS3, and BBS5). Both McKusick-Kaufman syndrome and Bardet-Biedl syndrome are inherited in an autosomal recessive pattern, and both syndromes are caused by mutations in the MKKS gene. However, mutations in MKKS are found in only 4%-11% of unselected Bardet-Biedl syndrome patients. We hypothesized that an analysis of patients with atypical Bardet-Biedl syndrome and McKusick-Kaufman syndrome (Group I; 15 probands) and patients with Bardet-Biedl syndrome who had linkage results inconsistent with linkage to the other loci (Group II; 12 probands) could increase the MKKS mutation yield. Both mutant alleles were identified in only two families in Group II. Single (heterozygous) sequence variations were found in three Group I families and in two Group II families. Combining these results with previously published data showed that only one mutant allele was detected in nearly half of all patients screened to date, suggesting that unusual mutational mechanisms or patterns of inheritance may be involved. However, sequencing of the BBS2 gene in these patients did not provide any evidence of digenic or "triallelic" inheritance. The frequency of detected mutations in MKKS in Group II patients was 24%, i.e., six times higher than the published rate for unselected BBS patients, suggesting that small-scale linkage analyses may be useful in suitable families.

Identification of the gene that, when mutated, causes the human obesity syndrome BBS4.

Bardet-Biedl syndrome (BBS, MIM 209900) is a heterogeneous autosomal recessive disorder characterized by obesity, pigmentary retinopathy, polydactyly, renal malformations, mental retardation, and hypogenitalism. The disorder is also associated with diabetes mellitus, hypertension, and congenital heart disease. Six distinct BBS loci map to 11q13 (BBS1), 16q21 (BBS2), 3p13-p12 (BBS3), 15q22.3-q23 (BBS4), 2q31 (BBS5), and 20p12 (BBS6). Although BBS is rare in the general population (<1/100,000), there is considerable interest in identifying the genes causing BBS because components of the phenotype, such as obesity and diabetes, are common. We and others have demonstrated that BBS6 is caused by mutations in the gene MKKS (refs. 12,13), mutation of which also causes McKusick-Kaufman syndrome (hydrometrocolpos, post-axial polydactyly, and congenital heart defects). MKKS has sequence homology to the alpha subunit of a prokaryotic chaperonin in the thermosome Thermoplasma acidophilum. We recently identified a novel gene that causes BBS2. The BBS2 protein has no significant similarity to other chaperonins or known proteins. Here we report the positional cloning and identification of mutations in BBS patients in a novel gene designated BBS4.

Positional cloning of a novel gene on chromosome 16q causing Bardet-Biedl syndrome (BBS2).

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous autosomal recessive disorder with the primary clinical features of obesity, pigmented retinopathy, polydactyly, hypogenitalism, mental retardation and renal anomalies. Associated features of the disorder include diabetes mellitus, hypertension and congenital heart disease. There are six known BBS loci, mapping to chromosomes 2, 3, 11, 15, 16 and 20. The BBS2 locus was initially mapped to an 18 cM interval on chromosome 16q21 with a large inbred Bedouin kindred. Further analysis of the Bedouin population allowed for the fine mapping of this locus to a 2 cM region distal to marker D16S408. Physical mapping and sequence analysis of this region resulted in the identification of a number of known genes and expressed sequence tag clusters. Mutation screening of a novel gene (BBS2) with a wide pattern of tissue expression revealed homozygous mutations in two inbred pedigrees, including the large Bedouin kindred used to initially identify the BBS2 locus. In addition, mutations were found in three of 18 unrelated BBS probands from small nuclear families.

A spectrum of FOXC1 mutations suggests gene dosage as a mechanism for developmental defects of the anterior chamber of the eye.

Mutations in the forkhead transcription-factor gene (FOXC1), have been shown to cause defects of the anterior chamber of the eye that are associated with developmental forms of glaucoma. Discovery of these mutations was greatly facilitated by the cloning and characterization of the 6p25 breakpoint in a patient with both congenital glaucoma and a balanced-translocation event involving chromosomes 6 and 13. Here we describe the identification of novel mutations in the FOXC1 gene in patients with anterior-chamber defects of the eye. We have detected nine new mutations (eight of which are novel) in the FOXC1 gene in patients with anterior-chamber eye defects. Of these mutations, five frameshift mutations predict loss of the forkhead domain, as a result of premature termination of translation. Of particular interest is the fact that two families have a duplication of 6p25, involving the FOXC1 gene. These data suggest that both FOXC1 haploinsufficiency and increased gene dosage can cause anterior-chamber defects of the eye.

Non-secretion of mutant proteins of the glaucoma gene myocilin in cultured trabecular meshwork cells and in aqueous humor.

Until recently, very little was known about the molecular mechanisms responsible for the development of glaucoma, a leading cause of blindness worldwide. Mutations in the glaucoma gene myocilin (MYOC, GLC1A) are associated with elevated intraocular pressure and the development of autosomal dominant juvenile glaucoma and a subset of adult-onset glaucoma. MYOC is expressed in the trabecular meshwork (TM), a tissue responsible for drainage of aqueous humor from the eye, and the tissue involved in elevated intraocular pressure associated with glaucoma. To better understand the role of MYOC in glaucoma pathogenesis, we examined the expression of normal and mutant myocilin in cultured ocular (TM) and non-ocular cells as well as in the aqueous humor of patients with and without MYOC glaucoma. Normal myocilin was secreted from cultured cells, but very little to no myocilin was secreted from cells expressing five different mutant forms of MYOC. In addition, no mutant myocilin was detected in the aqueous humor of patients harboring a nonsense MYOC mutation (Q368X). Co-transfection of cultured cells with normal and mutant myocilin led to suppression of normal myocilin secretion. These studies suggest that MYOC glaucoma is due either to insufficient levels of secreted myocilin or to compromised TM cell function caused by congestion of the TM secretory pathway.

Mutation of a nuclear receptor gene, NR2E3, causes enhanced S cone syndrome, a disorder of retinal cell fate.

Hereditary human retinal degenerative diseases usually affect the mature photoreceptor topography by reducing the number of cells through apoptosis, resulting in loss of visual function. Only one inherited retinal disease, the enhanced S-cone syndrome (ESCS), manifests a gain in function of photoreceptors. ESCS is an autosomal recessive retinopathy in which patients have an increased sensitivity to blue light; perception of blue light is mediated by what is normally the least populous cone photoreceptor subtype, the S (short wavelength, blue) cones. People with ESCS also suffer visual loss, with night blindness occurring from early in life, varying degrees of L (long, red)- and M (middle, green)-cone vision, and retinal degeneration. The altered ratio of S- to L/M-cone photoreceptor sensitivity in ESCS may be due to abnormal cone cell fate determination during retinal development. In 94% of a cohort of ESCS probands we found mutations in NR2E3 (also known as PNR), which encodes a retinal nuclear receptor recently discovered to be a ligand-dependent transcription factor. Expression of NR2E3 was limited to the outer nuclear layer of the human retina. Our results suggest that NR2E3 has a role in determining photoreceptor phenotype during human retinogenesis.

An autosomal genomic screen for autism. Collaborative linkage study of autism.

Autism is a severe neurodevelopmental disorder defined by social and communication deficits and ritualistic-repetitive behaviors that are detectable in early childhood. The etiology of idiopathic autism is strongly genetic, and oligogenic transmission is likely. The first stage of a two-stage genomic screen for autism was carried out by the Collaborative Linkage Study of Autism on individuals affected with autism from 75 families ascertained through an affected sib-pair. The strongest multipoint results were for regions on chromosomes 13 and 7. The highest maximum multipoint heterogeneity LOD (MMLS/het) score is 3.0 at D13S800 (approximately 55 cM from the telomere) under the recessive model, with an estimated 35% of families linked to this locus. The next highest peak is an MMLS/het score of 2.3 at 19 cM, between D13S217 and D13S1229. Our third highest MMLS/het score of 2.2 is on chromosome 7 and is consistent with the International Molecular Genetic Study of Autism Consortium report of a possible susceptibility locus somewhere within 7q31-33. These regions and others will be followed up in the second stage of our study by typing additional markers in both the original and a second set of identically ascertained autism families, which are currently being collected. By comparing results across a number of studies, we expect to be able to narrow our search for autism susceptibility genes to a small number of genomic regions. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 88:609-615, 1999.

Evaluation and molecular characterization of EHD1, a candidate gene for Bardet-Biedl syndrome 1 (BBS1).

Bardet-Biedl Syndrome (BBS) is an autosomal recessive disorder characterized by developmental abnormalities including mental retardation, obesity, retinitis pigmentosa, polydactyly, short stature, and hypogenitalism. To date, five BBS loci have been identified. BBS1, located on 11q13, is reported to be the most prevalent form of BBS in the Caucasian population. A positional cloning approach is being used to identify the gene responsible for BBS1. EHD1, a new member of the EH-domain containing proteins, was identified in this study as lying within the BBS1 disease interval. RNA analysis of many tissues revealed that expression of EHD1 is ubiquitous, with elevated levels in the testis. The genomic structure of EHD1 was elucidated by direct BAC sequencing. Following identification of the intron/exon boundaries, mutational analysis was performed by single strand conformation polymorphism and direct sequencing of affected individuals from several large kindreds linked to the BBS1 locus, as well as a cohort of unrelated probands. No disease-causing mutations were identified in this analysis, but several polymorphisms were found.

Expression of the Mf1 gene in developing mouse hearts: implication in the development of human congenital heart defects.

The transcription factor FKHL7 gene has recently been associated with the anterior segment dysgenesis disorder of the eye known as Axenfeld-Rieger anomaly (ARA). A growing body of evidence indicates that mutations in FKHL7 cause not only defects in the anterior segment of the eye but defects in the heart valves and septa as well. In order to evaluate its contribution to normal heart septation and valve formation, expression of the mouse homologue Mf1 in embryonic hearts was analyzed by in situ hybridization. A weak but significant level of Mf1 expression could be detected in the endocardium of mouse embryos as early as day 8.5 post-conception (p.c.). Mf1 expression was undetectable in the hearts of day 9.5 p.c. embryos, but by day 10.5-11 p.c., Mf1 transcripts could be found again in the endocardium of both the atrium and ventricle and a relatively strong signal was observed in the dorsal portion of the septum primum, in what appeared to be the spinal vestibule. At day 13 p.c. when aortic and pulmonary trunks are separated, relatively more Mf1 transcripts were detected in the leaflets of aortic, pulmonary, and venous valves, the ventral portion of the septum primum, as well as in the single layer of cells on the edges of the atrioventricular cushion tissues. Surprisingly, there was no signal detected in the developing interventricular septum. At day 15 p.c., overall Mf1 signals were greatly decreased. However, significant levels of expression could still be observed in the atrial septum, the tricuspid valve, the mitral valve, and in the venous valve but not in the interventricular septum. The temporal and spatial expression patterns of the Mf1 gene in developing mouse hearts suggest that Mf1 may play a critical role in the formation of valves and septa with the exception of the interventricular septum. This is further supported by our studies showing that mutations in the FKHL7 gene were associated with defects in the anterior segment of the eye as well as atrial septal defects or mitral valve defects. Dev Dyn 1999;216:16-27.

The cloning and developmental expression of unconventional myosin IXA (MYO9A) a gene in the Bardet-Biedl syndrome (BBS4) region at chromosome 15q22-q23.

Bardet-Biedl Syndrome (BBS) is a heterogeneous, autosomal recessive disorder characterized by mental retardation, obesity, retinitis pigmentosa, syndactyly and/or polydactyly, short stature, and hypogenitalism and is caused by mutations at a number of distinct loci. Using a positional cloning approach for identifying the BBS4 (chromosome 15) gene, we identified and cloned an unconventional myosin gene, myosin IXA (HGMW-approved symbol MYO9A). Since mutations in unconventional myosins are known to cause several human diseases, and since mutations of unconventional myosin VIIa cause retinal degeneration, we evaluated myosin IXA as a candidate for BBS. We exploited PCR-based techniques to clone a 8473-nt cDNA for myosin IXA. A 7644-bp open reading frame predicts a protein with all the hallmarks of class IX unconventional myosins. Human Northern blot analysis and in situ hybridization of mouse embryos reveal that myosin IXA is expressed in many tissues consistent with BBS. Intron/exon boundaries were identified, and myosin IXA DNA and RNA from BBS4 patients were evaluated for mutation.

The CMT2D locus: refined genetic position and construction of a bacterial clone-based physical map.

Charcot-Marie-Tooth (CMT) disease is a progressive neuropathy of the peripheral nervous system, typically characterized by muscle weakness of the distal limbs. CMT is noted for its genetic heterogeneity, with four distinct loci already identified for the axonal form of the disease (CMT2). In 1996, linkage analysis of a single large family revealed the presence of a CMT2 locus on chromosome 7p14 (designated CMT2D). Additional families have been linked subsequently to the same genomic region, including one with distal spinal muscular atrophy (dSMA) and one with mixed features of dSMA and CMT2; symptoms in both of these latter families closely resemble those seen in the original CMT2D family. There is thus a distinct possibility that CMT2 and dSMA encountered in these families reflect allelic heterogeneity at a single chromosome 7 locus. In the study reported here, we have performed more detailed linkage analysis of the original CMT2D family based on new knowledge of the physical locations of various genetic markers. The region containing the CMT2D gene, as defined by the original family, overlaps with those defined by at least two other families with CMT2 and/or dSMA symptoms. Both yeast artificial chromosome (YAC) and bacterial clone-based [bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC)] contig maps spanning approximately 3.4 Mb have been assembled across the combined CMT2D critical region, with the latter providing suitable clones for systematic sequencing of the interval. Preliminary analyses have already revealed at least 28 candidate genes and expressed-sequence tags (ESTs). The mapping information reported here in conjunction with the evolving sequence data should expedite the identification of the CMT2D/dSMA gene or genes.

The forkhead transcription factor gene FKHL7 is responsible for glaucoma phenotypes which map to 6p25.

A number of different eye disorders with the presence of early-onset glaucoma as a component of the phenotype have been mapped to human chromosome 6p25. These disorders have been postulated to be either allelic to each other or associated with a cluster of tightly linked genes. We have identified two primary congenital glaucoma (PCG) patients with chromosomal anomalies involving 6p25. In order to identify a gene involved in PCG, the chromosomal breakpoints in a patient with a balanced translocation between 6p25 and 13q22 were cloned. Cloning of the 6p25 breakpoint led to the identification of two candidate genes based on proximity to the breakpoint. One of these, FKHL7, encoding a forkhead transcription factor, is in close proximity to the breakpoint in the balanced translocation patient and is deleted in a second PCG patient with partial 6p monosomy. Furthermore, FKHL7 was found to harbour mutations in patients diagnosed with Rieger anomaly (RA), Axenfeld anomaly (AA) and iris hypoplasia (IH). This study demonstrates that mutations in FKHL7 cause a spectrum of glaucoma phenotypes.

Active generation glands present in neonates of some Cordylid Lizards: A case study of Cordylus macropholis (Sauria: Cordylidae).

Generation glands are holocrine epidermal glands occurring on the ventral aspect of the thigh of cordylid lizards. In most species these glands seemingly start to differentiate with the onset of sexual maturity, but macroscopic signs of generation gland activity were noted in neonates of the large-scaled girdled lizard, Cordylus macropholis. The glands of neonatal, subadult, and adult individuals were examined microscopically using standard histological techniques. The glands of the five neonatal specimens examined, including both males and females, all had the same basic structure and displayed two layers of mature glandular material. In subadult and adult specimens, the number of layers varied from seven to nine. The structure of the generation glands of C. macropholis is similar to that of the few other Cordylus species that have been described to date. They are of the protruding kind with multiple mature glandular generations. Juveniles of an additional 12 cordylid species have been examined for the presence of active generation glands. Active glands were found to be present in neonates of C. tasmani and C. tropidosternum, both of which are, like C. macropholis, terrestrial species. In C. cordylus and C. coeruleopunctatus, active generation glands are absent in neonates, but differentiate soon after birth. In other cordylid species, generation glands apparently differentiate only with the onset of sexual maturity. J. Morphol. 235:177-182, 1998. © 1998 Wiley-Liss, Inc.

Severe Charcot-Marie-Tooth neuropathy type 1A with 1-base pair deletion and frameshift mutation in the peripheral myelin protein 22 gene.

A 27-year-old man with negative family history and both parents with normal neurological evaluation and motor nerve conduction velocities (MNCVs) showed onset of severe weakness of feet at 4 years of age. Subsequently he developed left equinovarus deformity, thoracic scoliosis, ulnar nerve enlargement, areflexia, distal hypesthesia and slowing of MNCVs for median and ulnar nerves (15-25 m/sec). Molecular genetic studies showed deletion of one nucleotide (G330) (codon 94) in exon 3 of the PMP22 gene associated with frameshift mutation.

Dejerine-Sottas neuropathy in mother and son with same point mutation of PMP22 gene.

We studied a 25-year-old black woman with healthy parents and her 2-year, 11-month-old son. Her motor development was delayed and she started to walk with support when she was 6 years old. She never walked independently and had always used a wheelchair. Neurological evaluation showed severe weakness and atrophy of her feet, legs, and hands, bilateral pes cavus and hammertoes, corrected scoliosis, hypesthesia for proprioception and vibration sense in both feet and ankles, and areflexia. She had normal intelligence. Her son also had delayed motor milestones and was still unable to stand and walk independently at almost 3 years. Neurological evaluation revealed diffuse muscle hypotonia and weakness with generalized areflexia and normal intelligence. No muscle atrophies or feet deformities were noticed. Nerve conduction velocities showed significant slowing (less than 5 m/s) with prolonged distal latencies (above 30 ms). Compound motor action potential amplitudes were markedly reduced. Electromyography revealed polyphasic motor unit potentials. Molecular genetic studies indicated a Trembler type missense point mutation of exon 4 of the peripheral myelin protein 22 gene that led to the substitution of a spartic acid for glycine in both the mother and her son. Her parents showed normal DNA studies.

Dejerine-Sottas disease with sensorineural hearing loss, nystagmus, and peripheral facial nerve weakness: de novo dominant point mutation of the PMP22 gene.

A 32 year old woman with Dejerine-Sottas disease and negative family history is reported. Clinical onset of her condition was with congenital weakness of her distal four extremities, accompanied by peripheral facial nerve weakness, deafness, and nystagmus. She has used a wheelchair all her life. Sural nerve biopsy showed proliferation of Schwann cells, extensive endoneural fibrosis, axon loss, and demyelination. MNCVs showed marked slowing. MRI of the brain was normal. Molecular genetic studies indicated a de novo dominant missense point mutation of exon 3 of the peripheral myelin protein 22 gene at nucleotide 264 causing replacement of serine with leucine.

Autosomal dominant Charcot-Marie-Tooth axonal neuropathy mapped on chromosome 7p (CMT2D).

Clinical, electrophysiological and genetic linkage studies were performed on a large autosomal dominant family with Charcot-Marie-Tooth axonal neuropathy type 2 (CMT2) with 38 members of which 14 were affected. Onset of the disease was between 16 and 30 years of age with weakness and atrophy of the hands more severe than of the feet with slow progressive course in 12 patients. Deep tendon reflexes were absent in the upper extremities and decreased in the lower extremities. There was distal hypesthesia for touch, proprioception and vibration sense for the hands more than for the feet. Motor nerve conduction velocities showed normal values (48-53 M/s) with normal latencies (2-3 msec) and electromyography revealed signs of denervation. Genetic linkage analysis used 167 short tandem repeat markers (STRPs) spaced throughout the 22 autosomes. Linkage to the short arm of chromosome 7 at 7p14 was found using the marker D7S435 (Z = 4.83 at theta = 0). Flanking markers were D7S1808 and D7S1806 and the genetic distance between them was 6.8 cM. The multipoint linkage analysis gave a peek multipoint lod score of 6.89 between the markers D7S1808 and D7S435. Linkage analysis showed significantly negative lod scores (with values less than -2) with markers of chromosomes 1 and 3 where CMT axonal forms have been previously mapped. PFGE analysis indicated the absence of the CMT1A duplication. Our findings are consistent with a new genetic type of axonal CMT neuropathy designated by us as CMT2D. Potential candidate genes are multiple T-cell gamma receptor genes which map to the same cytogenetic interval as CMT2D neuropathy.

Mutations of the noncoding region of the connexin32 gene in X-linked dominant Charcot-Marie-Tooth neuropathy.

We studied two families with X-linked dominant Charcot-Marie-Tooth neuropathy. The clinical findings included onset around age 14 years, with moderate weakness of feet extensors and palmar and dorsal interossei, areflexia, distal hypesthesia, and slow progressivity. Motor nerve conduction velocities showed slowing (20 to 30 m/sec) and EMGs were normal. Genetic linkage analysis revealed positive lod scores with the markers of the Xq13.1 region in family 2, but was noninformative in family 1. There were no point mutations in the connexin32 gene coding region. Instead, family 1 revealed a T-to-G transversion at position -528 relative to the ATG start codon, whereas family 2 showed a C-to-T transition at position -458. The first mutation is located in the nerve-specific connexin32 promoter just upstream of the transcription start site, the second is located in the 5' untranslated region of the mRNA.

Correlation between connexin 32 gene mutations and clinical phenotype in X-linked dominant Charcot-Marie-Tooth neuropathy.

We studied the relationship between the genotype and clinical phenotype in 27 families with dominant X-linked Charcot-Marie-Tooth (CMTX1) neuropathy. Twenty-two families showed mutations in the coding region of the connexin32 (cx32) gene. The mutations include four nonsense mutations, eight missense mutations, two medium size deletions, and one insertion. Most missense mutations showed a mild clinical phenotype (five out of eight), whereas all nonsense mutations, the larger of the two deletions, and the insertion that produced frameshifts showed severe phenotypes. Five CMTX1 families with mild clinical phenotype showed no point mutations of the cx32 gene coding region. Three of these families showed positive genetic linkage with the markers of the Xq13.1 region. The genetic linkage of the remaining two families could not be evaluated because of their small size.

A Dejerine-Sottas neuropathy family with a gene mapped on chromosome 8.

The Patient is a 55-year-old black male who belongs to a large family with 9 affected relatives with autosomal dominant Dejerine-Sottas neuropathy (DSN). Onset of his condition was at 2 years of age with steppage gait followed by severe progressive weakness, atrophy, and sensory loss of his legs and hands accompanied by areflexia and thoracolumbar kyphoscoliosis. The patient became wheelchair confined at age 38. At around age 42, the left shoulder became dislocated and the humeral head underwent aseptic necrosis (Charcot joint). Nerve conduction studies showed absent motor and sensory responses for all major nerves tested. Genetic linkage suggested mapping of this DSN gene on chromosome 8qter. A younger brother with similar neurological findings also demonstrated Charcot joints with bone destruction of the joints of the fourth and fifth fingers.