Mapping of two new markers within the smallest interval harboring the spinal muscular atrophy locus by family and radiation hybrid analysis

The locus responsible for the childhood-onset proximal spinal muscular atrophies (SMA) has recently been mapped to an area of 2–3 Mb in the region q12–13.3 of chromosome 5. We have used a series of radiation hybrids (RHs) containing distinct parts of the SMA region as defined by reference markers. A cosmid library was constructed from one RH. Thirteen clones were isolated and five of these were mapped within the SMA region. Both RH mapping and fluorescence in situ hybridization analysis showed that two clones map in the region between loci D5S125 and D5S351. One of the cosmids contains expressed sequences. Polymorphic dinucleotide repeats were identified in both clones and used for segregation analysis of key recombinant SMA families. One recombination between the SMA locus and the new marker 9Ic (D5S685) indicates that 9Ic is probably the closest distal marker. The absence of recombination between the SMA locus and marker Fc (D5S684) suggests that Fc is located close to the disease gene. These new loci should refine linkage analysis in SMA family studies and may facilitate the isolation of the disease gene.     

Use of Genetic and Physical Mapping to Locate the Spinal Muscular Atrophy Locus between Two New Highly Polymorphic DNA Markers

The gene for autosomal recessive forms of spinal muscular atrophy (SMA) has recently been mapped to chromosome 5ql3, within a 4-cM region between the blocks D5S465/D5S125 and MAP-1B/D5S112. We identified two new highly polymorphic microsatellite DNA markers—namely, AFM265wf5 (D5S629) and AFM281yh9 (D5S637)—which are the closest markers to the SMA locus. Multilocus analysis by the location-score method was used to establish the best estimate of the SMA gene location. Our data suggest that the most likely location for SMA is between locus D5S629 and the block D5S637/D5S351/MAP-1B/D5S112/D5S357. Genetic analysis of inbred SMA families, based on homozygosity by descent and physical mapping using mega-YACs, gave additional information for the loci order as follows: cen–D5S6–D5S125/D5S465–D5S435–D5S629–SMA–D5S637–D5S351–MAP–1B/D5S112–D5S357–D5S39–tel. These data give the direction for bidirectional walking in order to clone this interval and isolate the SMA gene.     

Autistic Disorder and Chromosome 15q11–q13: Construction and Analysis of a BAC/PAC Contig

Autistic disorder (AD) is a neurodevelopmental disorder that affects approximately 2–10/10,000 individuals. Chromosome 15q11–q13 has been implicated in the genetic etiology of AD based on (1) cytogenetic abnormalities; (2) increased recombination frequency in this region in AD versus non-AD families; (3) suggested linkage with markers D15S156, D15S219, and D15S217; and (4) evidence for significant association with polymorphisms in the γ-aminobutyric acid receptor subunit B3 gene (GABRB3). To isolate the putative 15q11–q13 candidate AD gene, a genomic contig and physical map of the approximately 1.2-Mb region from the GABA receptor gene cluster to the OCA2 locus was generated. Twenty-one bacterial artificial chromosome (BAC) clones, 32 P1-derived artificial chromosome (PAC) clones, and 2 P1 clones have been isolated using the markers D15S540, GABRB3, GABRA5, GABRG3, D15S822, and D15S217, as well as 34 novel markers developed from the end sequences of BAC/PAC clones. In contrast to previous findings, the markers D15S822 and D15S975 have been localized within the GABRG3 gene, which we have shown to be approximately 250 kb in size. NotI and numerous EagI restriction enzyme cut sites were identified in this region. The BAC/PAC genomic contig can be utilized for the study of genomic structure and the identification and characterization of genes and their methylation status in this autism candidate gene region on human chromosome 15q11–q13.     

Two novel microsatellite markers for prenatal prediction of spinal muscular atrophy (SMA)

Autosomal recessive spinal muscular atrophy (SMA) has been mapped to a 6-cM interval on chromosome 5q12–13.3, flanked proximally by locus D5S6 and distally by locus D5S112. In this study we describe the isolation of two new microsatellite markers (EF1/2a and EF13/14) near locus D5S125, which lies 2 cM distal to D5S6. We show by linkage analysis and the study of the recombinants in 55 SMA pedigrees that the disease lies in the 4-cM interval between EF1/2a and D5S112. Fluorescence in situ analysis of cosmids from D5S6, EF1/2a and D5S112 confirms the genetic order and relative distance of markers. The microsatellites EF1/2a and EF13/14 are the first highly polymorphic PCR based proximal markers in SMA to be described, and will be of value in prental prediction of the disorder.     

Refined physical map of the spinal muscular atrophy gene (SMA) region at 5q13 based on YAC and cosmid contiguous arrays

The gene for the autosomal recessive neurodegenerative disorder spinal muscular atrophy has been mapped to a region of 5q13 flanked proximally by CMS-1 and distally by D5S557. We present a 2-Mb yeast artificial chromosome (YAC) contig constructed from three libraries encompassing the D5S435/D5S629/CMS-1—SMA—D5S557/D5S112 interval. The D5S629/CMS-1—SMA—D5S557 interval is unusual insofar as chromosome 5-specific repetitive sequences are present and many of the simple tandem repeats (STR) are located at multiple loci that are unstable in our YAC clones. A long-range restriction map that demonstrates the SMA-containing interval to be 550 kb is presented. Moreover, a 210-kb cosmid array from both a YAC-specific and a chromosome 5-specific cosmid library encompassing the multilocus STRs CATT-1, CMS-1, D5F149, D5F150, and D5F153 has been assembled. We have recently reported strong linkage disequilibrium with Type I SMA for two of these STRs, indicating that the gene is located in close proximity to or within our cosmid clone array.     

Genetic Analysis of the Epidermal Differentiation Complex (EDC) on Human Chromosome 1q21: Chromosomal Orientation, New Markers, and a 6-Mb YAC Contig

The epidermal differentiation complex (EDC) unites a remarkable number of structurally, functionally, and evolutionarily related genes that play an important role in terminal differentiation of the human epidermis. It is localized within 2.05 Mb of region q21 on human chromosome 1. We have identified and characterized 24 yeast artificial chromosome (YAC) clones by mapping individual EDC genes, sequence-tagged site (STS) markers (D1S305, D1S442, D1S498, D1S1664), and 10 new region-specific probes (D1S3619–D1S3628). Here we present a contig that covers about 6 Mb of 1q21 including the entire EDC. Fluorescencein situhybridization on metaphase chromosomes with two YACs flanking the EDC determined its chromosomal orientation and established, in conjunction with physical mapping results, the following order of genes and STSs: 1cen–D1S442–D1S498–S100A10–THH–FLG–D1S1664–IVL–SPRR3–SPRR1–SPRR2–LOR–S100A9–S100A8–S100A7–S100A6–S100A5–S100A4–S100A3–S100A2–S100A1–D1S305–1qtel. These integrated physical, cytogenetic, and genetic mapping data will be useful for linkage analyses of diseases associated with region 1q21 and for the identification of novel genes and regulatory elements in the EDC.     

Human Rod Monochromacy: Linkage Analysis and Mapping of a Cone Photoreceptor Expressed Candidate Gene on Chromosome 2q11

We have performed linkage analysis in eight families with rod monochromacy, an autosomal recessively inherited condition with complete color blindness. Significant linkage was found with markers located at the pericentromeric region of chromosome 2. A maximum lod score of 5.36 was obtained for marker D2S2333 at θ = 0.00. Mapping of meiotic breakpoints localized the disease gene between markers D2S2187 and D2S2229. Homozygosity for a number of subsequent markers indicating identity by descent was found in two families and provides evidence for a further refinement of the locus proximal to D2S373. This defines an interval of ≈3 cM covering theACHM2locus for rod monochromacy. Radiation hybrid mapping of theCNGA3gene encoding the α-subunit of the cGMP gated cation channel in human cone photoreceptors resulted in a maximum lod score of 16.1 with marker D2S2311 combined with a calculated physical distance of 6.19cR_10,000. Screening of the CEPH YAC library and subsequent STS mapping indicated the physical order cen–D2S2222–D2S2175–(D2S2187/D2S2311)–qtel ofmarkers on 2q11 and showed that theCNGA3gene maps most closely to D2S2187 and D2S2311. These data indicate that theCNGA3gene maps within the critical interval of theACHM2locus for rod monochromacy and thus is a candidate gene for this disease.     

No evidence for linkage of autosomal dominant proximal spinal muscular atrophies to chromosome 5q markers

Summary Two recent articles have reported the linkage of a gene for recessive spinal muscular atrophy (SMA) on the chromosome region 5q11.2–13.3. Our data show no linkage of the dominantly inherited forms of SMA to this chromosome region.     

High-Resolution Genetic Map of the Human Glutamyl Aminopeptidase Gene (ENPEP)

The murine B-lymphocyte differentiation antigen BP-1/6C3 has been identified as glutamyl aminopeptidase (EAP), the gene symbol for which isENPEP.Using genomic DNA encoding for human EAP as a probe, we identified theENPEPgene location on human chromosome 4q25 by polymerase chain reaction analysis of a human/rodent somatic cell hybrid mapping panel and by fluorescencein situhybridization. Using a radiation hybrid panel, the gene order aroundENPEPwas determined to be centromere–D4S1236–(570 kb)–ENPEP–(210 kb)–D4S262–(270 kb)–D4S953–(270 kb)–D4S474–(570 kb)–IF. The linkage ofENPEPto complement factor I (IF) confirms the human chromosome band 4q25 localization predicted from the chromosomal location of murineENPEP.HumanENPEPthus provides an additional marker for the long arm of chromosome 4 that should facilitate studies of this genomic region.     

A recombination event occurring within two complex 5q13.1 microsatellite repeat polymorphisms suggests a telomeric mapping of spinal muscular atrophy

The gene for the childhood spinal muscular atrophies (SMAs) has been mapped to 5q13.1. The interval containing the SMA gene has been defined by linkage analysis as 5qcen-D5S629-SMA-D5S557-5qter. We have identified a recombination event within this interval on a type-I SMA chromosome. The recombination maps to a region of multilocus microsatellite repeat (MSR) markers, and occurs between different subloci of two such markers, CMS-1 and 7613. While the possibility of a novel mutation caused by the recombination cannot be discounted, we believe when viewed in the context of a similar recombination in a Dutch SMA family, a centromeric boundary at the recombination site for the critical SMA interval is likely. This new proximal boundary would reduce the minimal region harboring the SMA locus from 1.1 Mb to approximately 600 kb.     

Long range restriction map of the von Hippel-Lindau gene region on human chromosome 3p

Von Hippel-Lindau disease is a heritable tumour syndrome caused by the loss of the function of a tumour suppressor gene on the short arm of human chromosome 3. The interval RAF1-D3S18 (3p25–3p26) has been identfied by genetic linkage studies to harbour the von Hippel-Lindau gene. We have constructed a long range restriction map of this region and have succeeded in demonstrating the physical linkage of loci D3S726 (DNA probe LIB31-38), D3S18 (c-LIB-1, L162E5), D3S601 (LIB1963) and D3S587 (LIB 12–48). Since multipoint analysis has located D3S601 proximal to D3S726, the physical map should be oriented with D3S726 towards the telomere. The order and distances of probes within the von Hippel-Lindau gene region is as follows: telomere — LIB3138 — (<280 kb) — c-LIB-1 — (overlapping) — L162E5 — (900–1600 kb) — (LIB 19-63, LIB 12–48) — centromere. In tissues that included blood, semen and Epstein-Barrvirus-transformed lymphocytes, we detected a putative CpG island flanking D3S18.     

Linkage analysis in spinal muscular atrophy, by six closely flanking markers on chromosome 5

The proximal spinal muscular atrophies (SMA) represent the second most common autosomal recessive disorder, after cystic fibrosis. The gene responsible for chronic SMA has recently been mapped to chromosome 5q by using genetic linkage studies. Among six markers mapping to this region, five were shown to be linked with the SMA locus in 39 chronic SMA families each containing at least two affected individuals. Multilocus analysis by the method of location score was used to establish the best estimate of the SMA gene location. Our data suggest that the most likely location for SMA is between loci D5S6 and D5S39. The genetic distances between these two markers are estimated to be 6.4 cM in males and 11.9 cM in females. Since meiosis were informative with D5S39 and D5S6 in 92% and 87% of SMA families, respectively, it is hoped that the present study will contribute to the calculation of genetic risk in SMA families.     

Subregional localization of the chromosome 21 loci D21S24 and D21S26 using physical mapping techniques

Summary We were able to refine the chromosomal position of two existing marker loci, using an extended chromosome 21 somatic cell hybrid panel. The locus D21S26 mapped in the region 21q11.2–q21.1, and the locus D21S24 in 21q22.1–q22.2. Physical and genetic analysis indicated that D21S26 is tightly linked to D21S13 and D21S16, two markers previously linked to familial Alzheimer's disease.     

Regional localization by in situ hybridization of a human chromosome 9 marker tightly linked to the Friedreich's ataxia locus

Summary In order to determine the regional localization of the Friedreich's ataxia (FA) gene on chromosome 9, the DNA probe DR47 (D9S5), which detects a restriction fragment length polymorphism (RFLP) in tight linkage with the disease, was hybridized in situ to metaphase chromosomes. Our results enable the D9S5 locus to be assigned to the 9q12–q13 region, thus indicating that this is also the localization of the FA gene.     

Identification of the Locus Associated with Diabetic Nephropathy in Type 1 Diabetes Mellitus

Polymorphic tetranucleotide microsatellites D3S1512, D3S1744, D3S1550, and D3S2326 were used to study the association of chromosome region 3q21–q25 neighboring the angiotensin II receptor type 1 gene (AT ₂ R1) with diabetic nephropathy (DN) in diabetes mellitus type 1 (DM1). Allele and genotype frequencies were compared for DM1 patients with (N = 39) or without (N = 62) DN. Fisher's exact test with Bonferroni's correction revealed significant differences in frequencies of two D3S2326 alleles, one D3S1512 allele, and one allele and one genotype of D3S1550. No significant difference was observed with D3S1744. Thus, region 3q21–q25 proved tightly associated with DN in ethnic Russians with DM1 from Moscow.     

Fine mapping of the Autosomal Dominant Split Hand/Split Foot Locus on Chromosome 7, Band q21.3-q22.1

Split hand/split foot (SHFD) is a human developmental defect characterized by missing digits, fusion of remaining digits, and a deep median cleft in the hands and feet. Cytogenetic studies of deletions and translocations associated with this disorder have indicated that an autosomal dominant split hand/split foot locus (gene SHFD1) maps to 7q21-q22. To characterize the SHFD1 locus, somatic cell hybrid lines were constructed from cytogenetically abnormal individuals with SHFD. Molecular analysis resulted in the localization of 93 DNA markers to one of 10 intervals surrounding the SHFD1 locus. The translocation breakpoints in four SHFD patients were encompassed by the smallest region of overlap among the SHFD-associated deletions. The order of DNA markers in the SHFD1 critical region has been defined as PON–D7S812–SHFD1–D7S811–ASNS. One DNA marker, D7S811, detected altered restriction enzyme fragments in three patients with translocations when examined by pulsed-field gel electro-phoresis (PFGE). These data map SHFD1, a gene that is crucial for human limb differentiation, to a small interval in the q21.3-q22.1 region of human chromosome 7.     

Genetic Linkage Mapping of the Dehydroepiandrosterone Sulfotransferase (STD) Gene on the Chromosome 19q13.3 Region

In the human liver and adrenal, there is a single hydroxysteroid sulfotransferase, which catalyzes the transformation of dehydroepiandrosterone to dehydroepiandrosterone sulfate, the most abundantly circulating steroid in humans, and also catalyzes the sulfation of a series of other 3β-hydroxysteroids as well as cholesterol. Dehydroepiandrosterone sulfate serves as precursor for the formation of active androgens and estrogens in several peripheral tissues, indicating that hydroxysteroid sulfotransferase plays a pivotal role in controlling the hormonal action of sex steroids by regulating their bioavailability. We recently elucidated the structure of the gene encoding hydroxysteroid sulfotransferase (STD), also designated dehydroepiandrosterone sulfotransferase, which spans 17 kb and contains six exons. The STD gene was preliminarily assigned to chromosome 19 by polymerase chain reaction (PCR) amplification of DNA from a panel of human/rodent somatic cell hybrids. To locate the STD gene, the novel biallelic polymorphism found in intron 2 was genotyped in eight CEPH reference families by direct sequencing of PCR products. Two-point linkage analysis was first performed between the latter polymorphism and chromosome 19 markers from Généthon and NIH/CEPH. The closest linkage was observed with D19S412 (Z_max= 9.23; θ_max0.038) and HRC (Z_max= 5.95; θ_max0.036), located on the 19q13.3 region. A framework map including six Généthon markers flanking the polymorphic STD gene was created by multipoint linkage analysis. Thereafter, a high-resolution genetic map of the region was constructed, yielding to the following order: qter–D19S414–D19S224–D19S420–D19S217–(APOC2–D19S412)–(STD–HRC)– KLK–D19S22–D19S180–PRKCG–D19S418–tel.     

Molecular characterization of a patient with del(1)(q23–q25)

Summary We report a patient (S.T.) with multiple congenital anomalies and developmental delay associated with an interstitial deletion of 1q23–1q25. Molecular analysis of the deletion was performed using DNA markers that map to 1q. Five DNA markers, MLAJ-1 (D1S61), CRI-L1054 (D1S42), HBI40 (D1S66), OS-6 (D1S75), and BH516 (D1S110), were demonstrated to be deleted. Informative polymorphisms demonstrated this to be a de novo deletion of the maternally derived chromosome. Deletion status was determined using restriction fragment length polymorphism (RFLP) analysis supplemented with densitometry in the experiments where RFLP analysis was not fully informative. Deletions were confirmed by Southern analysis using genomic DNA from a somatic cell hybrid retaining the del(1)(q23–q25) chromosome that was constructed from patient S.T. Flow karyotyping confirmed the deletion and estimated that the deletion encompassed 11,000–16,000 kb. The clinical and cytogenetic characteristics of S.T. are compared with those of ten previously described patients with monosomy 1q21–1q25.     

Association between Ag1-CA Alleles and Severity of Autosomal Recessive Proximal Spinal Muscular Atrophy

The gene for autosomal recessive proximal spinal muscular atrophy (SMA) has been mapped to an 850-kb interval on 5q11.2-q13.3, between the centromeric D5S823 and telomeric D5S557 markers. We report a new complex marker, Ag1-CA, that lies in this interval, whose primers produce one, two, or rarely three amplification-fragment-length variants (AFLVs) per allele. Class I chromosomes are those which amplify a single AFLV allele, and class II chromosomes are those which amplify an allele with two or three AFLVs. Ag1-CA shows highly significant allelic association with type I SMA in both the French Canadian (Hôpital Sainte-Justine [HSJ]) and American (Ohio State University [OSU]) populations (P<.0001). Significant association between the Ag1-CA genotype and disease severity was also observed. Type I patients were predominantly homozygous for class I chromosomes (P=.0003 OSU; P=.0012 HSJ), whereas the majority of type II patients were heterozygous for class I and II chromosomes (P=.0014 OSU; P=.001 HSJ). There was no significant difference in Ag1-CA genotype frequencies between type III patients (P=.5 OSU; P=.25 HSJ) and the paired normal chromosomes from both carrier parents. Our results indicate that Ag1-CA is the most closely linked marker to SMA and defines the critical candidate-gene region. Finally, we have proposed a model that should be taken into consideration when screening candidate SMA genes.