Faculty Bibliography

Autozygosity mapping in consanguineous families has proven to be a powerful method for identifying recessive disease genes. Using this technique with whole genome SNP data generated from low density mapping arrays, we previously identified two genes that underlie autosomal recessive woolly hair (ARWH/hypotrichosis; OMIM278150), specifically P2RY5 and Lipase H (LIPH). In the current study, we sought to identify a novel disease locus for ARWH/hypotrichosis by analyzing two large consanguineous families from Pakistan who had initially been excluded for mutations at either of these disease loci by haplotype analysis with microsatellite markers. A genome-wide analysis of 10 members from each of the two families failed to identify significant regions of autozygosity or linkage. Upon genotyping an additional 10 family members in one of the families, parametric linkage analysis identified a region on chromosome 3q27 with evidence for linkage (Z = 2.5). Surprisingly, this region contains the LIPH gene. Microsatellite markers located within the LIPH gene were used for haplotype analysis and demonstrated that not one, but two haplotypes were segregating with the phenotype in each of these families. DNA sequencing identified two distinct LIPH mutations (280_369dup90 and 659_660delTA). Each affected individual (n = 38) was either homozygous for one mutation (n = 7 and 16 respectively), or compound heterozygous (n = 15). A review of the literature identified several reports of compound heterozygotes in consanguineous families. Prompted by this finding, we derived the probability that a patient affected with a recessive disease is carrying two mutations at the disease locus. We suggest that the validity of the IBD assumption may be challenged in large consanguineous families.

While there have been significant advances in understanding the genetic etiology of human hair loss over the previous decade, there remain a number of hereditary disorders for which a causative gene has yet to be identified. We studied a large, consanguineous Brazilian family that presented with woolly hair at birth that progressed to severe hypotrichosis by the age of 5, in which 6 of the 14 offspring were affected. After exclusion of known candidate genes, a genome-wide scan was performed to identify the disease locus. Autozygosity mapping revealed a highly significant region of extended homozygosity (lod score of 10.41) that contained a haplotype with a linkage lod score of 3.28. Results of these two methods defined a 9-Mb region on chromosome 13q14.11-q14.2. The interval contains the P2RY5 gene, in which we recently identified pathogenic mutations in several families of Pakistani origin affected with autosomal recessive woolly and sparse hair. After the exclusion of several other candidate genes, we sequenced the P2RY5 gene and identified a homozygous mutation (C278Y) in all affected individuals in this family. Our findings show that mutations in P2RY5 display variable expressivity, underlying both hypotrichosis and woolly hair, and underscore the essential role of P2RY5 in the tissue integrity and maintenance of the hair follicle.

The genetic determinants of hair texture in humans are largely unknown. Several human syndromes exist in which woolly hair comprises a part of the phenotype; however, simple autosomal recessive inheritance of isolated woolly hair has only rarely been reported. To identify a gene involved in controlling hair texture, we performed genetic linkage analysis in six families of Pakistani origin with autosomal recessive woolly hair (ARWH; OMIM 278150). All six families showed linkage to chromosome 13q14.2-14.3 (Z = 17.97). In all cases, we discovered pathogenic mutations in P2RY5, which encodes a G protein-coupled receptor and is a nested gene residing within intron 17 of the retinoblastoma 1 (RB1) gene. P2RY5 is expressed in both Henle's and Huxley's layers of the inner root sheath of the hair follicle. Our findings indicate that disruption of P2RY5 underlies ARWH and, more broadly, uncover a new gene involved in determining hair texture in humans.

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.

Plasma non-HDL and HDL cholesterol levels are predictors of cardiovascular diseases. We carried out a genetic cross between two laboratory inbred mouse strains, C57BL/6J and CASA/Rk, to detect loci that control the plasma levels of non-HDL and HDL cholesterol. With regard to non-HDL cholesterol, chow-fed CASA/Rk males and females had 87% and 25% higher levels, respectively, than did C57BL/6Js. The levels of non-HDL cholesterol in F1s were similar to C57BL/6J. There was no strain difference in HDL cholesterol levels. An intercross between F1s was performed, and plasma non-HDL and HDL cholesterol was measured in 185 male and 184 female mice. In both male and female F2 mice, plasma non-HDL and HDL cholesterol levels were unimodally distributed; however, in both cases the values for females were significantly lower than for males. Therefore, linkage analysis was performed with sex as a covariate. Significant linkage for non-HDL cholesterol was found on chromosome 6 at 49 cM (LOD 5.17), chromosome 4 at 55 cM (LOD 4.22), and chromosome 8 at 7 cM (LOD 3.68). Significant linkage for HDL cholesterol was found on chromosome 9 at 14 cM (LOD 7.52) and chromosome 8 at 76 cM (LOD 4.69). A significant epistatic interaction involving loci on chromosomes 2 and 5 was also observed for non-HDL cholesterol. In summary, linkage analysis in these cross-identified novel loci confirmed previously identified loci in control of plasma non-HDL and HDL cholesterol and disclosed a novel interaction in controlling non-HDL cholesterol levels in the mouse.

A problem associated with automated analysis of fluorescently labeled fragments separated by slab gel or capillary electrophoresis is the doublet peak formed when Taq DNA Polymerase adds a nontemplated nucleotide (generally an adenosine) to the 3' end of the product.This nontemplated addition (plus A) is primarily dependent on the 5' sequence of the reverse primer and, to a lesser extent, polymerase chain reaction (PCR) conditions. Primers may amplify the true product, the plus A product or a doublet product comprised of both. When using markers based on dinucleotide repeats, this single base pair difference can make binning and accurate automated analysis problematic. To drive the PCR reaction consistently to the plus A product, the sequence of the nonfluorescent primer used in amplification can be modified by adding a 5' tail favoring the nontemplated addition. The present study, conducted by the Fragment Analysis Research Group (FARG) of the Association of Biomolecular Resource Facilities, provided researchers with an opportunity to compare normal products amplified with a dinucleotide marker to products amplified with the same primer to which a 5' tail designed to promote the plus A product had been added. The study also included a sample amplified with a tetranucleotide repeat marker for comparison. The results from this study were returned to the FARG for comprehensive analysis and are reported here.