In Silico Analysis of Missense Mutations in LPAR6 Reveals Abnormal Phospholipid Signaling Pathway Leading to Hypotrichosis

Autosomal recessive hypotrichosis is a rare genetic irreversible hair loss disorder characterized by sparse scalp hair, sparse to absent eyebrows and eyelashes, and sparse axillary and body hair. The study, presented here, established genetic linkage in four families showing similar phenotypes to lysophosphatidic acid receptor 6 (LPAR6) gene on chromosome 13q14.11-q21.32. Subsequently, sequence analysis of the gene revealed two previously reported missense mutations including p.D63V in affected members of one and p.I188F in three other families. Molecular modeling and docking analysis was performed to investigate binding of a ligand oleoyl-L-alpha-lysophosphatidic acid (LPA) to modeled protein structures of normal and mutated (D63V, G146R, I188F, N248Y, S3T, L277P) LPAR6 receptors. The mutant receptors showed a complete shift in orientation of LPA at the binding site. In addition, hydropathy analysis revealed a significant change in the membrane spanning topology of LPAR6 helical segments. The present study further substantiated involvement of LPAR6-LPA signaling in the pathogenesis of hypotrichosis/woolly hair and provided additional insight into the molecular mechanism of hair development.     

Novel mutations in G protein-coupled receptor gene (P2RY5) in families with autosomal recessive hypotrichosis (LAH3)

Autosomal recessive hypotrichosis (LAH3) is a rare hair disorder characterized by sparse hair on scalp and the rest of the body of affected individuals. Recently mutations in a G protein-coupled receptor gene, P2RY5, located at LAH3 locus, have been reported in several families with autosomal recessive hypotrichosis simplex and woolly hair. For the present study, 22 Pakistani families with autosomal recessive hypotrichosis were enrolled. Genotyping using microsatellite markers linked to three autosomal recessive forms of hypotrichosis (LAH1, LAH2, LAH3) showed the linkage of 2 families to the LAH2 locus and 14 to the LAH3 locus. The remaining 6 families were not linked to any of the three loci. Families linked to LAH3 locus were further subjected to screening of the P2RY5 gene with direct DNA sequencing. Three previously reported variants, c.69insCATG (p.24insHfs52), c.188A > T (p.D63V) and c.565G > A (p.E189K) were observed in eight families. Four novel nonsynonymous sequence variants, c.8G > C (p.S3T), c.36insA (p.D13RfsX16), c.160insA (p.N54TfsX58) and c.436G > A (p.G146R) were found to segregate within six families. Z. Azeem, M. Jelani, G. Naz, M. Tariq, N. Wasif, S. Kamran-ul-Hassan Naqvi contributed equally to this work.     

Genetic mapping of a novel hypotrichosis locus to chromosome 7p21.3–p22.3 in a Pakistani family and screening of the candidate genes

Hereditary hypotrichosis is a heterogeneous group of inherited hair loss disorders characterized by diffused or localized thinning or absence of hair affecting scalp, eyebrows and eyelashes, and other body parts. Over the past few years, at least four autosomal dominant and six autosomal recessive forms of hypotrichosis have been described. All these ten forms of hypotrichosis have been mapped on different human chromosomes and the corresponding genes have been identified in most of these cases. In the present study, we have described a six-generation Pakistani consanguineous family with an autosomal recessive transmission of hereditary hypotrichosis. All the five affected individuals of the family showed complete absence of scalp hair and sparse eyebrows and eyelashes. They were born with complete absence of scalp hairs. Facial hair of beard and mustaches were present in all the affected adult male individuals. Papules were observed only on scalp of the affected individuals. A scalp biopsy from an affected individual showed markedly reduced number of hair follicles. Human genome scan using polymorphic microsatellite markers mapped the disease locus on chromosome 7p21.3–p22.3, flanked by markers D7S1532 and D7S3047. A maximum two-point LOD score of 4.74 (θ = 0.00) was obtained at marker D7S481. The linkage interval spans 15.69 cM, which corresponds to 6.59 Mb according to the sequence-based physical map (Build 36.2). Mutation analysis of five potential candidate genes (GNA12, FOXK1, DAGLB, ZNF12, ACTB), located in the linkage interval, did not reveal any functional sequence variant.     

Novel mutations in the keratin-74 (KRT74) gene underlie autosomal dominant woolly hair/hypotrichosis in Pakistani families

Autosomal dominant woolly hair (ADWH) is an inherited condition of tightly curled and twisted scalp hair. Recently, a mutation in human keratin-74 (KRT74) gene has been shown to cause this form of hereditary hair disorder. In the present study, we have described two families (A and B) having multiple individuals affected with autosomal dominant form of hair loss disorders. In family A, 10 individuals showed ADWH phenotype while in the family B, 14 individuals showed hypotrichosis of the scalp. Genotyping using polymorphic microsatellite markers showed linkage of both the families to type II keratin gene cluster on the chromosome 12q12-14.1. Mutation analysis of the KRT74 gene identified two novel mutations in the affected individuals of the families. The sequence analysis revealed a splice acceptor site mutation (c.IVS8-1G>A) in family A and a missense variant (c.1444G>A, p.Asp482Asn) in family B. Mutations identified in the present study extend the body of evidence implicating the KRT74 gene in the pathogenesis of autosomal dominant hair loss disorders.     

Genomewide scan for linkage reveals evidence of several susceptibility loci for alopecia areata

Alopecia areata (AA) is a genetically determined, immune-mediated disorder of the hair follicle that affects 1%-2% of the U.S. population. It is defined by a spectrum of severity that ranges from patchy localized hair loss on the scalp to the complete absence of hair everywhere on the body. In an effort to define the genetic basis of AA, we performed a genomewide search for linkage in 20 families with AA consisting of 102 affected and 118 unaffected individuals from the United States and Israel. Our analysis revealed evidence of at least four susceptibility loci on chromosomes 6, 10, 16 and 18, by use of several different statistical approaches. Fine-mapping analysis with additional families yielded a maximum multipoint LOD score of 3.93 on chromosome 18, a two-point affected sib pair (ASP) LOD score of 3.11 on chromosome 16, several ASP LOD scores >2.00 on chromosome 6q, and a haplotype-based relative risk LOD of 2.00 on chromosome 6p (in the major histocompatibility complex locus). Our findings confirm previous studies of association of the human leukocyte antigen locus with human AA, as well as the C3H-HeJ mouse model for AA. Interestingly, the major loci on chromosomes 16 and 18 coincide with loci for psoriasis reported elsewhere. These results suggest that these regions may harbor gene(s) involved in a number of different skin and hair disorders.     

The gene CYP3 encoding P450pcn1 (nifedipine oxidase) is tightly linked to the gene COL1A2 encoding collagen type 1 alpha on 7q21-q22.1.

CYP3, the gene which encodes the hepatic cytochrome P450pcn1, the isozyme responsible for the metabolic oxidation of the calcium channel-blocking drug nifedipine, has recently been mapped to human chromosome 7 using somatic cell hybrids. Using multilocus linkage analysis in CEPH families, we examined the linkage of a cDNA probe (hPCN1) for CYP3 to the oncogene MET, the pro-alpha 2(1) collagen gene COL1A2, and the T-cell receptor beta-chain gene TCRB, together with three arbitrary loci D7S8, D7S13, and D7S16, defined by the anonymous DNA probes pJ3.11, pB79a, and p7C22, respectively. From 70 CEPH parents screened with a StyI RFLP for hPCN1, four informative families were found each with both parental and maternal grandparents and 6-11 children per family. Tight linkage emerged between CYP3 and COL1A2, with a maximum combined lod score of 5.72 at theta = 0, suggesting the most likely subchromosomal localization of CYP3 is 7q21.3-q22.1.     

Vitiligo: complex segregation and linkage disequilibrium analyses with respect to microsatellite loci spanning the HLA

Familial clustering and linkage disequilibrium studies suggest that genetic factors predispose to vitiligo, although a clear transmission pattern and cosegregation of vitiligo with specific mutations have not been demonstrated. We collected pedigree data on vitiligo from a set of 56 multigeneration families belonging to the Paisa community from Antioquia, Colombia, with the goal of applying the unified model of complex segregation and linkage disequilibrium analyses to test the hypotheses of the existence of a major gene predisposing to vitiligo and that allelic or haplotype polymorphisms of microsatellite loci at 6p21.3-21.4 spanning HLA (D6S276, D6S265, D6S273, and D6S291) are associated with this predisposition. Minimum sibship sample size to discriminate dominant and recessive inheritance models was largely accomplished. Between the 15 models of complex segregation used, the one that best fitted the data was that of a major dominant gene and the existence of strong environmental effects acting on the recessive genotype. The penetrance and risk estimations discriminated two sets of vitiligo patients: those with early onset of vitiligo cosegregating with a dominant mode of inheritance without environmental effects, and those with late onset of vitiligo cosegregating with the recessive genotype and being influenced by environmental effects. After establishing the normal distribution of allelic frequencies and performing multiple comparisons correction, the linkage disequilibrium analysis suggested that a major genetic factor could be located at 6p21.3-21.4, because we detected significant case-control differences for allele 122 at D6S265 ( Pc=0.0264) and significant linkage disequilibrium between loci D6S276 and D6S273 in the cases but not in the controls. We cannot explain these results as a consequence of evolutionary forces or as genetic stratification acting differentially on cases and controls, because there was neither deviation from the Hardy-Weinberg expectations nor genetic subdivision between cases and controls, as theta; (non-biased F(ST)) was not significantly different from 0.     

Genome-wide scan and fine-mapping linkage study of androgenetic alopecia reveals a locus on chromosome 3q26

Androgenetic alopecia (AGA, male pattern baldness) is the most common form of hair loss. The origin of AGA is genetic, with the X chromosome located androgen receptor gene (AR) being the only risk gene identified to date. We present the results of a genome-wide linkage study of 95 families and linkage fine mapping of the 3q21-q29, 11q14-q25, 18p11-q23, and 19p13-q13 regions in an extended sample of 125 families of German descent. The locus with strongest evidence for linkage was mapped to 3q26 with a nonparametric linkage (NPL) score of 3.97 (empirical p value = 0.00055). This is the first step toward the identification of new susceptibility genes in AGA, a process which will provide important insights into the molecular and cellular basis of scalp hair loss.     

Allelic association of microsatellites of 6p in Italian hemochromatosis patients

Hemochromatosis (HC) is an inherited disorder of iron metabolism and is frequently seen in Caucasians. The biochemical defect and the responsible gene are unknown, but the HC locus is closely linked to HLA-A on human chromosome 6 in the region 6p21.3. Although extensive studies have been performed in several populations, the precise location of the gene is still undefined. Linkage disequilibrium with HC has been detected for loci that are 3 cM apart: HLA class I and D6S105, which is located on the telomeric side of HLA-A. We have analyzed the inheritance of several multi-allele polymorphisms that map to 6p (D6S265, Y52, HLA-F, D6S306, D6S105, D6S464, D6S299) in 34 Italian HC families and in 17 unrelated patients. Significant association with HC was shown for alleles of multiple markers in the HLA-A region, for the distant marker D6S105, but not for the D6S299 marker at 4 cM from HLA-A on the telomeric side. HC status was unambiguously assigned to 70 affected and 63 unaffected chromosomes from family studies. Thirty five different haplotypes were found in 70 HC chromosomes when considering four markers most tighly associated with the disease. A predominant haplotype comprising alleles 1-3-1-8 (marker order D6S265, HLA-A, Y52, D6S105) accounted for 30% of the HC chromosomes and was absent in normals. A minority of other HC haplotypes could be related to the major haplotype by assuming single crossover events. Results of haplotype studies suggest a founder effect in the Italian population, as previously shown in Australian patients, and a possible common mutation shared with affected individuals of Celtic origin. Received: 16 May 1995 / Revised 21 August 1995     

A region of primer binding variation at the D6S265 locus associated with HLA-A25 and HLA-A26 antigens

D6S265 is a polymorphic dinucleotide repeat, mapped within 70 kb centromeric of HLA-A, on chromosome 6p21.3. While genotyping families for genetic linkage analysis, allele non-amplification resulting in apparent non-Mendelian inheritance was observed at the D6S265 locus in 15 individuals, on chromosomes carrying the HLA-A25 and HLA-A26 antigens. The D6S265 locus was sequenced in a variant individual homozygous for allele non-amplification, and in a non-HLA-A25/-A26 individual, homozygous for D6S265 allele 1. Five base changes were identified in the reverse primer binding region of the variant individual, effectively preventing annealing of the 3 primer to the template.     

Evidence that a locus for familial high myopia maps to chromosome 18p.

Myopia, or nearsightedness, is the most common human eye disorder. A genomewide screen was conducted to map the gene(s) associated with high, early-onset, autosomal dominant myopia. Eight families that each included two or more individuals with >=-6.00 diopters (D) myopia, in two or more successive generations, were identified. Myopic individuals had no clinical evidence of connective-tissue abnormalities, and the average age at diagnosis of myopia was 6.8 years. The average spherical component refractive error for the affected individuals was -9.48 D. The families contained 82 individuals; of these, DNA was available for 71 (37 affected). Markers flanking or intragenic to the genes for Stickler syndrome types 1 and 2 (chromosomes 12q13.1-q13.3 and 6p21.3, respectively), Marfan syndrome (chromosome 15q21.1), and juvenile glaucoma (chromosome 1q21-q31) were also analyzed. No evidence of linkage was found for markers for the Stickler syndrome types 1 and 2, the Marfan syndrome, or the juvenile glaucoma loci. After a genomewide search, evidence of significant linkage was found on chromosome 18p. The maximum LOD score was 9.59, with marker D18S481, at a recombination fraction of .0010. Haplotype analysis further refined this myopia locus to a 7.6-cM interval between markers D18S59 and D18S1138 on 18p11.31.     

Birt-Hogg-Dubé syndrome, a genodermatosis associated with spontaneous pneumothorax and kidney neoplasia, maps to chromosome 17p11.2

Birt-Hogg-Dubé syndrome (BHD), an inherited autosomal genodermatosis characterized by benign tumors of the hair follicle, has been associated with renal neoplasia, lung cysts, and spontaneous pneumothorax. To identify the BHD locus, we recruited families with cutaneous lesions and associated phenotypic features of the BHD syndrome. We performed a genomewide scan in one large kindred with BHD and, by linkage analysis, localized the gene locus to the pericentromeric region of chromosome 17p, with a LOD score of 4.98 at D17S740 (recombination fraction 0). Two-point linkage analysis of eight additional families with BHD produced a maximum LOD score of 16.06 at D17S2196. Haplotype analysis identified critical recombinants and defined the minimal region of nonrecombination as being within a <4-cM distance between D17S1857 and D17S805. One additional family, which had histologically proved fibrofolliculomas, did not show evidence of linkage to chromosome 17p, suggesting genetic heterogeneity for BHD. The BHD locus lies within chromosomal band 17p11.2, a genomic region that, because of the presence of low-copy-number repeat elements, is unstable and that is associated with a number of diseases. Identification of the gene for BHD may reveal a new genetic locus responsible for renal neoplasia and for lung and hair-follicle developmental defects.     

Cervical carcinoma progression-associated genetic alterations on chromosome 6

To identify the loci associated with progression of cervical carcinoma, chromosome 6 regions were tested for loss of heterozygosity. Detailed analysis with 28 microsatellite markers revealed a high frequency of allelic deletions for several loci of the short (6p25, 6p22, 6p21.3) and long (6q14, 6q16-21, 6q23-24, 6q25, 6q27) arms of chromosome 6. Examination of 37 microdissected carcinoma and 22 cervical dysplasia specimens revealed allelic deletions from the HLA class I-III genes (6p22-21.3) and subtelomeric locus 6p25 were found in more than 40% dysplasia specimens. With multiple microdissection of cryosections, genetic heterogeneity of squamous cervical carcinoma was analyzed, and clonal and subclonal allelic deletions from chromosome 6 were identified. Half of the tumors had clonal allelic deletion of D6S273 (6p21.3), which is in a Ly6G6D (MEGT1) intron in the HLA class III gene locus. The frequency of allelic deletions from the chromosome 6 long arm was no more than 20% in dysplasias. Allelic deletions from two loci, 6q14 and 6q16-21, were for the first time associated with invasion and metastasis in cervical carcinoma.     

A gene for universal congenital alopecia maps to chromosome 8p21-22.

Complete or partial congenital absence of hair (congenital alopecia) may occur either in isolation or with associated defects. The majority of families with isolated congenital alopecia has been reported to follow an autosomal-recessive mode of inheritance (MIM 203655). As yet, no gene has been linked to isolated congenital alopecia, nor has linkage been established to a specific region of the genome. In an attempt to map the gene for the autosomal recessive form of the disorder, we have performed genetic linkage analysis on a large inbred Pakistani family in which affected persons show complete absence of hair development (universal congenital alopecia). We have analyzed individuals of this family, using >175 microsatellite polymorphic markers of the human genome. A maximum LOD score of 7.90 at a recombination fraction of 0 has been obtained with locus D8S258. Haplotype analysis of recombination events localized the disease to a 15-cM region between marker loci D8S261 and D8S1771. We have thus mapped the gene for this hereditary form of isolated congenital alopecia to a locus on chromosome 8p21-22 (ALUNC [alopecia universalis congenitalis]). This will aid future identification of the responsible gene, which will be extremely useful for the understanding of the biochemistry of hair development.     

Genetic homogeneity in Sjögren-Larsson syndrome: linkage to chromosome 17p in families of different non-Swedish ethnic origins.

Sjögren-Larsson syndrome (SLS) is a rare, autosomal recessive disorder that is characterized by congenital ichthyosis, mental retardation, and spastic diplegia or tetraplegia. Three United States families, three Egyptian families, and one Israeli Arab family were investigated for linkage of the SLS gene to a region of chromosome 17. Pairwise and multipoint linkage analysis with nine markers mapped the SLS gene to the same region of the genome as that reported in Swedish SLS pedigrees. Examination of recombinants by haplotype analysis showed that the gene lies in the region containing the markers D17S953, D17S805, D17S689, and D17S842. D17S805 is pericentromeric on 17p. Patients in two consanguineous Egyptian families were homozygous at the nine marker loci tested, and another patient from a third family was homozygous for eight of the nine, suggesting that within each of these families the region of chromosome 17 carrying the SLS gene is identical by descent. Linkage of the SLS gene to chromosome 17p in families of Arabic, mixed European, Native American, and Swedish descent provides evidence for a single SLS locus and should prove useful for diagnosis and carrier detection in worldwide cases.     

A gene for autosomal dominant hypohidrotic ectodermal dysplasia (EDA3) maps to chromosome 2q11-q13.

Autosomal dominant hypohidrotic ectodermal dysplasia (ADHED) is a disorder characterized by fine, slow-growing scalp and body hair, sparse eyebrows and eyelashes, decreased sweating, hypodontia, and nail anomalies. By genetic linkage analysis of a large ADHED kindred, we have mapped a gene for ADHED (EDA3) to the proximal long arm of chromosome 2 (q11-q13). Obligate recombinations localize EDA3 to an approximately 9-cM interval between D2S1321 and D2S308, with no apparent recombinations with markers D2S1343, D2S436, D2S293, D2S1894, D2S1784, D2S1890, D2S274, and CHLC.GAAT11C03.     

Regional mapping of the gene for autosomal dominant spinocerebellar ataxia (SCA1) by localizing the closely linked D6S89 locus to 6p24.2----p23.05.

The locus for one subtype of autosomal dominant spinocerebellar ataxias (SCA1) is closely linked (within 1-2 cM) to D6S89, which contains a highly polymorphic dinucleotide repeat sequence. D6S89 has been mapped previously to 6p24----p21.3, between the HLA and F13A1 loci. Mutant cell lines were used to correlate the absence or presence of D6S89 with cytogenetically detectable interstitial 6p deletions. The results allowed us to map D6S89 to the 6p24.2----p23.05 region. The close linkage of SCA1 to D6S89 indicates that this locus is most likely located in the 6p24----p23 segment.     

Localization of the hemochromatosis gene close to D6S105.

The hemochromatosis (HC) gene is known to be linked to HLA-A (6p21.3); however, its precise location has been difficult to determine because of a lack of additional highly polymorphic markers for this region. The recent identification of short tandem repeat sequences (microsatellites) has now provided this area with a number of markers with similar polymorphic index to the HLA serological polymorphisms. Using four microsatellites--D6S105, D6S109, D6S89, and F13A--together with the HLA class I loci HLA-A and HLA-B in 13 large pedigrees clearly segregating for HC, we have been able to refine the location of the HC gene. We identified no recombination between HC and HLA-A or D6S105, and two-point analyses placed the HC gene within one centimorgan (cM) of HLA-A and D6S105 (HLA-A maximum of the lod score [Zmax] of 9.90 at recombination fraction [theta] of 0.0, and D6S105 Zmax of 8.26 at theta of 0.0). The markers HLA-B, D6S109, D6S89, and F13A were separated from the HC locus by recombination, defining the centromeric and telomeric limits for the HC gene as HLA-B and D6S109, respectively. A multipoint map constructed using HLA-B, HLA-A, and D6S109 indicates that the HC gene is located in a region less than 1 cM proximal to HLA-A and less than 1 cM telomeric of HLA-A. These pedigree data indicate an association between HC and specific alleles at HLA-A and D6S105 (i.e., HLA-A3 and D6S105 allele 8).(ABSTRACT TRUNCATED AT 250 WORDS)     

Loss of heterozygosity at chromosome 6 as a marker of early genetic alterations in cervical intraepithelial neoplasias and microinvasive carcinomas

Oncogenic human papillomaviruses (mostly HPV types 16 and 18) are the major cause of cervical intraepithelial neoplasia (CIN), which progresses into cervical cancer (CC). To reveal early genetic alterations of chromosome 6 that are important for CC progression, we analyzed the loss of heterozygosity (LOH) in DNAs from 45 CIN cases, 47 microcarcinomas, and 19 invasive squamous cell carcinomas stage IB. LOH analysis of DNA samples prepared with microdissection from all CIN foci, as well as from CC lesions and synchronous CIN, permitted investigation of CIN and CC heterogeneity. Out of all CC stage I cases, 79% showed LOH with six microsatellite markers at chromosome 6. LOH with the microsatellite markers D6S276 (6p22) and TNFa (6p21.3) was found in 50% of the CC cases. LOH frequency in CIN lesions synchronous with CC was higher then in CIN cases without cancer; the statistical significance (P = 0.004) was shown for D6S291 (6p21.2). The finding suggests that the high frequency of LOH in CIN lesions is a marker of unfavorable prognosis for CIN. Progression from microcarcinoma to invasive CC of stage IB was associated with a higher LOH frequency at D6S344 (6p25) and TNFa (6p21.3). Early genetic alterations were found in CIN with microsatellites D6S273 and TNFa located at 6p21.3. Moreover, LOH frequency at D6S273 remained the same in both CIN and CC cases. Based on HPV typing, LOH analysis, and X-chromosome inactivation, the polyclonality of CC lesions, as well as CIN, was observed in a few patients.     

The Severe Perinatal Form of Autosomal Recessive Polycystic Kidney Disease Maps to Chromosome 6p21.1-p12: Implications for Genetic Counseling

Autosomal recessive polycystic kidney disease (ARPKD) is a one of the most common hereditary renal cystic diseases in children. Its clinical spectrum is widely variable with most cases presenting in infancy. Most affected neonates die within the first few hours of life. At present, prenatal diagnosis relies on fetal sonography, which is often imprecise in detecting even the severe form of the disease. Recently, in a cohort of families with mostly milder ARPKD phenotypes, an ARPKD locus was mapped to a 13-cM region of chromosome 6p21-cen. To determine whether severe perinatal ARPKD also maps to chromosome 6p, we have analyzed the segregation of seven microsatellite markers from the ARPKD interval in 22 families with the severe phenotype. In the majority of the affected infants, ARPKD was documented by histopathology. Our data confirm linkage and refine the ARPKD region to a 3.8-cM interval, delimited by the markers D6S465/D6S427/D6S436/D6S272 and D6S466. Taken together, these results suggest that, despite the wide variability in clinical phenotypes, there is a single ARPKD gene. These linkage data and the absence of genetic heterogeneity in all families tested to date have important implications for DNA-based prenatal diagnoses as well as for the isolation of the ARPKD gene.