Increased symptoms of attention deficit hyperactivity disorder and major depressive disorder symptoms in Nail-patella syndrome: potential association with LMX1B loss-of-function.

Nail-Patella syndrome (NPS) is an autosomal dominant disorder that is the result of heterozygous loss-of-function mutations in LMX1B, coding for a LIM homeobox (LIM-HD) transcription factor. Analyses of lmx1b mutant mice have revealed the role of Lmx1b in the development of mesencephalic dopaminergic neurons and the serotonergic system; these areas have been linked with symptoms of attention deficit hyperactivity disorder (ADHD) and major depressive disorder (MDD). Fifty adults (38 females, 12 males) with NPS completed the Conners' Adult ADHD Rating Scales-Self-report: Long Version (CAARS) and Beck Depression Inventory-II (BDI-II). The objective was to describe the neurobehavioral phenotype of these subjects and examine possible relationships between neurobehavioral symptoms and NPS. Elevated levels of DSM-IV-TR ADHD Inattentive symptoms were reported on the CAARS by 22% of the NPS sample. The BDI-II Total score was elevated for 40% of the NPS sample. There was a significant increase in the odds of an elevated BDI-II Total score when any of the three CAARS scales were elevated (odds ratios ranging from 11.455 to 15.615). The CAARS and BDI-II did not significantly differ with gender, age, or education level. There was no significant association between genetic mutation-predicted protein status and elevations on CAARS or BDI-II. Individuals with NPS reported co-occurring symptoms of ADHD and MDD, with higher levels of co-occurrence than reported in the literature for the general population. The co-occurrence of these symptoms may be related to mesencephalic dopaminergic neurologic pathway abnormalities that are a consequence of LMX1B loss of function.

Albinism and developmental delay: the need to test for 15q11-q13 deletion.

We report on a 17-month-old African girl with cutaneous and ophthalmologic features of oculocutaneous albinism type 2 as well as microcephaly, absent speech, and tremulous movements. Mutations of the P gene within the Angelman/Prader-Willi syndrome critical region at 15q11-q13 cause oculocutaneous albinism type 2. Comorbid oculocutaneous albinism and Angelman syndrome were suspected and confirmed by cytogenetics. Phenotypic features of Angelman syndrome or Prader-Willi syndrome in a patient with albinism should prompt further investigation.

Skeletal integrity in patients with nail patella syndrome.

Nail patella syndrome (NPS) is a rare autosomal dominant disorder resulting from a heterogeneous loss of function in the LMX1B gene. It is associated with multiple skeletal deformities, yet it is unknown whether this is associated with osteoporosis. To examine bone mass and the prevalence of fragility fractures, we assessed bone mineral density (BMD) of the spine and hip in 31 adults and 12 children with mutation-confirmed NPS and 60 healthy age- and gender-matched adult controls. For the adults with NPS, BMD was 11-20% lower at the hip sites (P < or = 0.001) and 8% lower at the spine (P < 0.05) than that of controls. Even when adjusted for body mass index, the BMD remained significantly lower in patients with NPS in all hip regions but not in the spine. Adults with NPS also had a significantly lower Z-score (sd values from normal) at all hip sites (all P < 0.05), compared with age- and gender-matched controls in the manufacturer's database. However, children had significantly lower Z-scores only at the femoral neck and trochanter. Participants with NPS also had a higher prevalence of fractures (odds ratio 30.9, 95% confidence interval 6.4-149.6, P < 0.001) and scoliosis (odds ratio 16.0, 95% confidence interval 3.3-78.2, P < 0.001). The majority of these fractures occurred in women before puberty and in long bones, especially the clavicle. We conclude that adults with NPS have a BMD that is 8-20% lower than controls, which is associated with an increase in the prevalence of fractures and scoliosis. Future studies are needed to determine whether bone quality, geometry, or turnover could account for these findings.

A neurological phenotype in nail patella syndrome (NPS) patients illuminated by studies of murine Lmx1b expression.

Nail patella syndrome (NPS) is an autosomal dominant disorder affecting development of the limb, kidney and eye. NPS is the result of heterozygous loss-of-function mutations in the LIM-homeodomain transcription factor, LMX1B. Recent studies suggest that the NPS phenotype may be more extensive than recognized previously including neurologic and neurobehavioral aspects. To determine whether these findings correlated with the expression of Lmx1b during development, an internal ribosomal entry site-LacZ reporter was inserted into the 3'UTR of the endogenous murine gene. The pattern of Lmx1b expression during the development of the limb, eye and kidney correlates with the NPS phenotype. Additional sites of expression were observed in the central nervous system (CNS). The effects of the absence of Lmx1b in the CNS were determined in lmx1b-/- mice by histology and immunocytochemistry. Lmx1b is required for the differentiation and migration of neurons within the dorsal spinal cord. The inability of afferent sensory neurons to migrate into the dorsal horn is entirely consistent with diminished pain responses in NPS patients.

Evidence for linkage of nonsyndromic cleft lip with or without cleft palate to a region on chromosome 2.

Results from a genome-wide screen of 10 multiplex families ascertained through probands with nonsyndromic cleft lip with or without cleft palate (CL/P) in Mexico, Argentina, and the United States yielded suggestive evidence of linkage to chromosomes 2, 6, 17 and 18. Fine mapping excluded all regions except chromosome 2. Subsequent analysis was performed on the original 10 families plus an additional 16 families using 31 markers on chromosome 2. This analysis showed intriguing evidence of linkage to 2q (Zlr=2.26, empirical P-value=0.028 in a chromosome-wide analysis). Transmission disequilibrium tests also revealed evidence of linkage and disequilibrium for two markers in this region (D2S168 and D2S1400 with P-values=0.022 and 0.006, respectively). A subset of these 26 families provided additional evidence for a susceptibility gene for CL/P on 2q, suggesting that further studies of genes in this region are warranted.

Association between genes on chromosome 4p16 and non-syndromic oral clefts in four populations.

Isolated cleft lip with or without cleft palate and cleft palate are among the most common human birth defects. Several candidate gene studies on MSX1 have shown significant association between markers in MSX1 and risk of oral clefts, and re-sequencing studies have identified multiple mutations in MSX1 in a small minority of cases, which may account for 1-2% of all isolated oral clefts cases. We explored the 2-Mb region around MSX1, using a marker map of 393 single nucleotide polymorphisms (SNPs) in 297 cleft lip, with or without cleft palate, case-parent trios and 84 cleft palate trios from Maryland, Taiwan, Singapore, and Korea. Both individual markers and haplotypes of two to five SNPs showed several regions yielding statistical evidence for linkage and disequilibrium. Two genes (STK32B and EVC) yielded consistent evidence from cleft lip, with or without cleft palate, trios in all four populations. These two genes plus EVC2 also yielded suggestive evidence for linkage and disequilibrium among cleft palate trios. This analysis suggests that several genes, not just MSX1, in this region may influence risk of oral clefts.

Association between IRF6 and nonsyndromic cleft lip with or without cleft palate in four populations.

PURPOSE: The interferon regulatory factor 6 (IRF6), the gene that causes van der Woude syndrome has been shown to be associated with nonsyndromic cleft lip with or without palate in several populations. This study aimed to confirm the contribution of IRF6 to cleft lip with or without palate risk in additional Asian populations. METHODS: A set of 13 single nucleotide polymorphisms was tested for association with cleft lip with or without palate in 77 European American, 146 Taiwanese, 34 Singaporean, and 40 Korean case-parent trios using both the transmission disequilibrium test and conditional logistic regression models. RESULTS: Evidence of linkage and association was observed among all four populations; and two specific haplotypes [GC composed of rs2235373-rs2235371 (p.V274I) and AAG of rs599021-rs2235373-rs595918] showed the most significant over- and undertransmission among Taiwanese cases (P=9x10(-6) and P=5x10(-6), respectively). The AGC/CGC diplotype composed of rs599021-rs2235373-rs2013162 showed almost a 7-fold increase in risk among the Taiwanese sample (P<10(-3)). These results confirmed the contribution of this gene to susceptibility of oral clefts across different populations; however, the specific single nucleotide polymorphisms showing statistical significance differed among ethnic groups. CONCLUSION: The high-risk genotypes and diplotypes identified here may provide a better understanding of the etiological role of this gene in oral clefts and potential options for genetic counseling.

Family-based analysis of MSX1 haplotypes for association with oral clefts.

Oral clefts, one of the most common forms of birth defects, are considered to be of complex etiology, including both genetic and environmental causes. To date, however, no particular genetic cause has been confirmed for isolated, nonsyndromic oral clefts. Previous case-control and family-based association studies reported an association between an intronic CA repeat of the MSX1 gene and risk for oral clefts. In this study, we identify eight single-nucleotide polymorphisms (SNPs) in the MSX1 gene, and present genotype results for these SNPs in a set of 206 oral cleft cases and their parents. We performed single-marker and haplotype-based transmission disequilibrium tests (TDTs), and tested for evidence of interaction between MSX1 haplotypes and exposure to maternal smoking in the first trimester, using a case-only approach. The haplotype TDT analyses further implicate this gene, or region, in controlling the risk for oral clefts, particularly for cleft palate. In addition, case-only haplotype analyses suggest an interaction between variation in the MSX1 gene and exposure to maternal smoking. This study encourages further focus on the MSX1 gene region to ultimately determine specific variants predisposing to oral clefts.

Association of human aging with a functional variant of klotho.

Mice deficient in Klotho gene expression exhibit a syndrome resembling premature human aging. To determine whether variation in the human KLOTHO locus contributes to survival, we applied two newly characterized polymorphic microsatellite markers flanking the gene in a population-based association study. In a cohort chosen for its homogeneity, Bohemian Czechs, we demonstrated significant differences in selected marker allele frequencies between newborn and elderly individuals (P < 0.05). These results precipitated a search for functional variants of klotho. We identified an allele, termed KL-VS, containing six sequence variants in complete linkage disequilibrium, two of which result in amino acid substitutions F352V and C370S. Homozygous elderly individuals were underrepresented in three distinct populations: Bohemian Czechs, Baltimore Caucasians, and Baltimore African-Americans [combined odds ratio (OR) = 2.59, P < 0.0023]. In a transient transfection assay, secreted levels of klotho harboring V352 are reduced 6-fold, whereas extracellular levels of the S370 form are increased 2.9-fold. The V352/S370 double mutant exhibits an intermediate phenotype (1.6-fold increase), providing a rare example of intragenic complementation in cis by human single nucleotide polymorphisms. The remarkable conservation of F352 among homologous proteins suggests that it is functionally important. The corresponding substitution, F289V, in the closest human klotho paralog with a known substrate, cBGL1, completely eliminates its ability to cleave p-nitrophenyl-beta-D-glucoside. These results suggest that the KL-VS allele influences the trafficking and catalytic activity of klotho, and that variation in klotho function contributes to heterogeneity in the onset and severity of human age-related phenotypes.

The human LMX1B gene: transcription unit, promoter, and pathogenic mutations.

LMX1B is a LIM-homeodomain transcription factor required for the normal development of dorsal limb structures, the glomerular basement membrane, the anterior segment of the eye, and dopaminergic and serotonergic neurons. Heterozygous loss-of-function mutations in LMX1B cause nail patella syndrome (NPS). To further understand LMX1B gene regulation and to identify pathogenic mutations within the coding region, a detailed analysis of LMX1B gene structure was undertaken. 5' -RACE and primer extension identified a long 5' -untranslated region of 1.3 kb that contains two upstream open-reading frames (uORFs). Transient transfection assays showed that sequences required for basal promoter activity extend no further than 112 bp upstream. An additional 47 mutations have been identified in the coding region, as well as nine deletions of large portions of the gene, but not in the promoter or highly conserved intronic sequences. The range of mutations and the identification of uORFs suggest further complexity in the regulation of LMX1B expression.

A genome-wide scan for loci predisposing to non-syndromic cleft lip with or without cleft palate in two large Syrian families.

Non-syndromic cleft lip with/without cleft palate (CL/P) is a common, usually non-fatal birth defect of complex etiology. Several segregation analyses have demonstrated that genetic factors are important in the pathogenesis of CL/P, most likely through the interaction of several genes of modest effects. The aim of this study was to perform a genome-wide linkage analysis to identify/search for candidate gene loci for CL/P. We conducted a genome-wide search in two large, relatively isolated Syrian families, each one with a large number of cases with CL/P (18 in family 1 and 4 in family 2). A locus with a multipoint LOD score of 2.80 and a 2-point non-parametric MLS LOD of 3.0 was detected on 17p13.1. Other chromosomal regions with multipoint LOD scores > or = 1.2 (P < or = 0.01) included 3p21.2, 4q32.1, and 7q34. These data indicate the possible presence of several susceptibility loci for CL/P and identify a strong candidate locus for this common birth defect on chromosome 17p13.

QRX, a novel homeobox gene, modulates photoreceptor gene expression.

A novel paired-like homeobox gene, designated as Qrx, was identified by a yeast one-hybrid screen using the bovine Rhodopsin promoter Ret-1 DNA regulatory element as bait. Qrx is preferentially expressed in both the outer and inner nuclear layers of the retina. Its homeodomain is nearly identical to that of Rx/Rax, a transcription factor that is essential for eye development, but it shares only limited homology elsewhere. Although Qrx and Rx/Rax show similar DNA binding properties in vitro, the two proteins demonstrate distinct target selectivity and functional behavior in promoter activity assays. QRX synergistically increases the transactivating function of the photoreceptor transcription factors Crx and NRL and it physically interacts with CRX. Qrx is present in the bovine and human genomes, but appears to be absent from the mouse genome. Nonetheless, a 5.8 kb upstream region of human QRX is capable of directing expression in presumptive photoreceptor precursor cells in transgenic mice. These results indicate that Qrx may be involved in modulating photoreceptor gene expression. In addition, the finding of rare heterozygous QRX sequence changes in three individuals with retinal degeneration raises the possibility that QRX may be involved in disease pathogenesis.

Meta-analysis of 13 genome scans reveals multiple cleft lip/palate genes with novel loci on 9q21 and 2q32-35.

Isolated or nonsyndromic cleft lip with or without cleft palate (CL/P) is a common birth defect with a complex etiology. A 10-cM genome scan of 388 extended multiplex families with CL/P from seven diverse populations (2,551 genotyped individuals) revealed CL/P genes in six chromosomal regions, including a novel region at 9q21 (heterogeneity LOD score [HLOD]=6.6). In addition, meta-analyses with the addition of results from 186 more families (six populations; 1,033 genotyped individuals) showed genomewide significance for 10 more regions, including another novel region at 2q32-35 (P=.0004). These are the first genomewide significant linkage results ever reported for CL/P, and they represent an unprecedented demonstration of the power of linkage analysis to detect multiple genes simultaneously for a complex disorder.