Jannine D. Cody

Genetic Linkage of Paget Disease of the Bone to Chromosome 18q

Paget disease is a common bone disease characterized by abnormal osteoclasts that are large, multinucleated, and overactive and that contain paramyxovirus-like nuclear inclusions. There is evidence for a major genetic component to Paget disease, with up to 40% of patients having affected first-degree relatives; however, the locus (loci) and gene(s) involved are unknown. Another bone disorder, familial expansile osteolysis (FEO), although extremely rare, also is characterized by similar osteoclast abnormalities but has an earlier age at onset and a more aggressive clinical progression. The causative gene for FEO has been localized to a region of human chromosome 18q. On the basis of the presence of similar clinical findings and of viral-like nuclear inclusions in osteoclasts, we hypothesized that FEO and Paget disease are allelic versions of the same locus. Therefore, a large kindred with a high incidence of Paget disease was examined to determine if Paget disease was linked to genetic markers in the same region of chromosome 18 as that for FEO. Our analysis yielded a two-point LOD score of 3.40, with the genetic marker D18S42, a marker tightly linked to the FEO locus. This demonstrates that the gene(s) responsible for FEO and that for Paget disease are either closely linked or the same locus.

Three novel mutations in SQSTM1 identified in familial Paget's disease of bone.

Mutations in Sequestosome 1 (SQSTM1) have been shown to segregate with familial Pagets disease of bone (PDB). We examined the coding sequence of SQSTM1 in five PDB pedigrees and found three novel mutations clustered around the C‐terminal ubiquitin associated domain. Disruptions of the C‐terminal domain of SQSTM1 seem to be a leading cause of familial PDB.

Congenital anomalies and anthropometry of 42 individuals with deletions of chromosome 18q.

Deletions of chromosome 18q are among the most common segmental aneusomies compatible with life. The estimated frequency is approximately 1/40,000 live births [Cody JD, Pierce JF, Brkanac Z, Plaetke R, Ghidoni PD, Kaye CI, Leach RJ. 1997. Am. J. Med. Genet. 69:280–286]. Most deletions are terminal encompassing as much as 36 Mb, but interstitial deletions have also been reported. We have evaluated 42 subjects with deletions of 18q at our institution. This is the largest number of individuals with this chromosome abnormality studied by one group of investigators. Here we report the physical findings in these individuals. We have compared our findings with those of previously reported cases and have found a significantly different incidence of several minor anomalies in our subjects. We also describe here several anomalies not previously reported in individuals with deletions of 18q, including short frenulum, short palpebral fissures, disproportionate short stature, overlap of second and third toes, and a prominent abdominal venous pattern. Characteristics found in subjects were analyzed for correlation with cytogenetic breakpoints. Several traits were found to correlate with the extent of the deletion. Large deletions were associated with significantly decreased head circumference and ear length as well as the presence of proximally placed and/or anomalous thumbs. Individuals with the smallest deletions were more likely to have metatarsus adductus. Although relatively few genotype/phenotype correlations were apparent, these data demonstrate that correlations with breakpoint are possible. This implies that more correlations will become evident when the more precise molecularly based genotyping is completed. These correlations will identify critical regions on the chromosome in which genes responsible for specific abnormal phenotypes are located. Am. J. Med. Genet. 85:455–462, 1999.

Magnetic Resonance Imaging Demonstrates Incomplete Myelination in 18q- Syndrome: Evidence for Myelin Basic Protein Haploinsufficiency

Magnetic resonance imaging (MRI) and MRI relaxometry were used to investigate disturbed brain myelination in 18q- syndrome, a disorder characterized by mental retardation, dysmorphic features, and growth failure. T1-weighted and dual spin-echo T2-weighted MR images were obtained, and T1 and T2 parametric image maps were created for 20 patients and 12 controls. MRI demonstrated abnormal brain white matter in all patients. White matter T1 and T2 relaxation times were significantly prolonged in patients compared to controls at all ages studied, suggesting incomplete myelination. Chromosome analysis using fluorescence in situ hybridization techniques showed that all patients with abnormal MRI scans and prolonged white matter T1 and T2 relaxation times were missing one copy of the myelin basic protein (MBP) gene. The one patient with normal-appearing white matter and normal white matter T1 and T2 relaxation times possessed two copies of the MBP gene. MRI and molecular genetic data suggest that incomplete cerebral myelination in 18q- is associated with haploinsufficiency of the gene for MBP.

Preferential Loss of the Paternal Alleles in the 18q- Syndrome

Individuals with the 18q- syndrome have variable deletions from the long arm of chromosome 18. They also exhibit a highly variable phenotype. To correlate genotype with phenotype accurately, extensive molecular and phenotypic analyses are needed on each affected individual. As a part of this analysis, we have determined the parental origin of the deleted chromosome in 34 individuals with the 18q- syndrome. We have found that 85% of the de novo deletions are paternal in origin. The percentage of fathers of individuals with paternally derived deletions who were > 30 years old was (not significantly) greater than that of the general population. The mothers of individuals with maternally derived deletions were near an average age for childbearing compared to the general population. Individuals with maternally derived terminal deletions had breakpoints as varied as those with paternally derived deletions. These results are consistent with the hypothesis that the reduced incidence of maternally derived deletions is not due to reduced viability, since individuals with large maternally derived deletions of chromosome 18q were found. We hypothesize that the prevalence of paternally derived deletions is due to an increased frequency of chromosome breakage in male germ cells. These results are consistent with results observed in other segmental aneusomies in which there is a high incidence of paternally derived deletions.

Identification of Cryptic Rearrangements in Patients with 18q− Deletion Syndrome

The majority of patients with 18q- syndrome appear cytogenetically to have a terminal deletion of the long arm of chromosome 18. These 18q- patients are diagnosed by use of standard cytogenetic banding techniques, which have resolution insufficient for precise genotyping. In our effort to obtain a thorough genotype, we have analyzed the DNA from 35 patients who originally were diagnosed as having de novo terminal deletions of chromosome 18. Molecular analysis was performed with polymorphic markers throughout the 18q- region. Cytogenetic FISH was performed with two human 18q telomeric probes, a chromosome 18-specific alpha-satellite probe, and whole chromosome 18-specific paint. Of 35 patients previously reported to have terminal deletions of 18q, we found that 5 (14%) have more-complex cryptic rearrangements and that 3 (9%) retain the most distal portion of 18q, consistent with an interstitial rather than a terminal deletion. These findings indicate that a standard karyotype can lead to insufficient characterization in 18q- syndrome. This has important ramifications for phenotype mapping of this syndrome, as well as for proper prognosis.

Growth Hormone Insufficiency Associated With Haploinsufficiency at 18q23

Growth hormone insufficiency is a common cause of growth failure in children with the 18q- syndrome. Individuals with this syndrome have a deletion as large as 36 Mb from the long arm of chromosome 18. We have evaluated 33 children with this syndrome for growth hormone production and have identified a region of approximately 2 Mb, which is deleted in every growth hormone insufficient patient. Two genes contained in this region, myelin basic protein, and the galanin receptor, are candidate genes for the growth hormone insufficiency phenotype.

Mapping Structural Differences of the Corpus Callosum in Individuals With 18q Deletions Using Targetless Regional Spatial Normalization

Individuals with a constitutional chromosome abnormality consisting of a deletion of a portion of the long arm of chromosome 18 (18q−) have a high incidence (∼95%) of dysmyelination. Neuroradiologic findings in affected children report a smaller corpus callosum, but this finding has not been quantified. This is in part due to the large intersubject variability of the corpus callosum size and shape and the small number of subjects with 18q−, which leads to low statistical power for comparison with typically developing children. An analysis method called targetless spatial normalization (TSN) was used to improve the sensitivity of statistical testing. TSN converges all images in a group into what is referred as group common space. The group common space conserves common shape, size, and orientation while reducing intragroup variability. TSN in conjunction with a Witelson vertical partitioning scheme was used to assess differences in corpus callosum size between 12 children with 18q− and 12 age‐matched normal controls. Significant global and regional differences in corpus callosum size were seen. The 18q− group showed an overall smaller (25%) corpus callosum (P < 10−7), even after correction for differences in brain size. Regionally, the posterior portions of corpus callosum (posterior midbody, isthmus, and splenium), which contain heavily myelinated fibers, were found to be 25% smaller in the population with 18q−. Hum Brain Mapping 24:325–331, 2005.

Molecular characterization of a patient with central nervous system dysmyelination and cryptic unbalanced translocation between chromosomes 4q and 18q.

We report on a 12‐year‐old boy who presented with delayed development and CNS dysmyelination. Genetic studies showed a normal 46,XY karyotype by routine cytogenetic analysis, and 46,XY.ish del(18)(q23)(D18Z1+, MBP−) by FISH using a locus‐specific probe for the MBP gene (18q23). Though the patient appeared to have normal chromosome 18s by repeated high resolution banding analysis, his clinical features were suggestive of a deletion of 18q. These included hearing loss secondary to stenosis of the external auditory canals, abnormal facial features, and foot deformities. FISH studies with genomic probes from 18q22.3 to 18qter confirmed a cryptic deletion which encompassed the MBP gene. In an attempt to further characterize the deletion, whole genome screening was conducted using array based comparative genomic hybridization (array CGH) analysis. The array CGH data not only confirmed a cryptic deletion in the 18q22.3 to 18qter region of approximately 7 Mb, it also showed a previously undetected 3.7 Mb gain of 4q material. FISH studies demonstrated that the gained 4q material was translocated distal to the 18qter deletion breakpoint. The 18q deletion contains, in addition to MBP, other known genes including CYB5, ZNF236, GALR1, and NFATC1, while the gained 4q material includes the genes FACL1 and 2, KLKB1, F11 and MTNR1A. The use of these combined methodologies has resulted in the first reported case in which array CGH has been used to characterize a congenital chromosomal abnormality, highlighting the need for innovative molecular cytogenetic techniques in the diagnosis of patients with idiopathic neurological abnormalities.

High resolution genomic analysis of 18q− using oligo-microarray comparative genomic hybridization (aCGH)†

The advent of oligonucleotide array comparative genomic hybridization (aCGH) has revolutionized diagnosis of chromosome abnormalities in the genetics clinic. This new technology also has valuable potential as a research tool to investigate larger genomic rearrangements that are typically diagnosed via routine karyotype. aCGH was used as a tool for the high‐resolution analysis of chromosome content in individuals with known deletions of chromosome 18. The aim of this study was to clarify the precise location of the breakpoints as well as to determine the presence of occult translocations creating additional deletions and duplications. One hundred eighty‐nine DNA samples from individuals with 18q deletions were analyzed. No breakpoint clusters were identified, as no more than two individuals had breakpoints within 2 kb of each other. Only two regions of 18q were never found to be haploid, suggesting the existence of haplolethal genes in those regions. Of the individuals with only a chromosome 18 abnormality, 17% (n = 29) had interstitial deletions. Six percent (n = 11) had a region of duplication immediately proximal to the deletion. Eight percent (n = 15) had more complex rearrangements with captured (non‐18q) telomeres thus creating a trisomic region. The 15 captured telomeres originated from a limited number of other telomeres (4q, 10q, 17p, 18p, 20q, and Xq). These data were converted into a format for ease of viewing and analysis by creating custom tracks for the UCSC Genome Browser. Taken together, these findings confirm a higher level of variability and genomic complexity surrounding deletions of 18q than has previously been appreciated.