Mind the Quality Gap When Banking on Dry Blood Spots.

Dry blood spots (DBS) offer many advantages over other blood banking protocols due to the reduction of time and equipment needed for collection and the ease of processing, storage, and shipment. In addition, the sample size makes it a very attractive method when considering the banking of small pediatric samples. On that note, the Centers for Disease Control and Prevention (CDC) preanalytical standards for DBS are commonly used in the worldwide mass spectrometry-based inborn errors of metabolism screening programs. However, these guidelines may not apply for analytes and protocols not included in these programs. In fact, the availability of leftover samples and the ongoing interest in protocols outside this scenario are providing us with new DBS biobanking insights. Herein, we review the literature for indicators that should be considered in the design of prospective fit for purpose DBS biobanks, especially for those focused mostly on pediatric and OMIC platforms.

Genome-wide association identifies SKIV2L and MYRIP as protective factors for age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly in the developed world. We conducted a genome-wide association study in a series of families enriched for AMD and completed a meta-analysis of this new data with results from reanalysis of an existing study of a late-stage case-control cohort. We tested the top findings for replication in 1896 cases and 1866 controls and identified two novel genetic protective factors for AMD. In addition to the complement factor H (CFH) (P=2.3 × 10⁻64) and age-related maculopathy susceptibility 2 (ARMS2) (P=1.2 × 10⁻6°) loci, we observed a protective effect at rs429608, an intronic SNP in SKIV2L (P=5.3 × 10⁻¹5), a gene near the complement component 2 (C2)/complement factor B (BF) locus, that indicates the protective effect may be mediated by variants other than the C2/BF variants previously studied. Haplotype analysis at this locus identified three protective haplotypes defined by the rs429608 protective allele. We also identified a new potentially protective effect at rs2679798 in MYRIP (P=2.9 × 10⁻4), a gene involved in retinal pigment epithelium melanosome trafficking. Interestingly, MYRIP was initially identified in the family-based scan and was confirmed in the case-control set. From these efforts, we report the identification of two novel protective factors for AMD and confirm the previously known associations at CFH, ARMS2 and C3.

Complement factor H polymorphisms, renal phenotypes and age-related macular degeneration: the Blue Mountains Eye Study.

Complement factor H (CFH) is a key regulator of the alternative pathway of complement and its mutations have been associated with membranoproliferative glomerulonephritis type II, atypical hemolytic uremic syndrome and age-related macular degeneration (AMD), suggesting that alternative pathway dysregulation is a common pathogenetic feature of these ocular and renal conditions. In this study we tested the hypothesis that common CFH variants have a global role in renal function in the Australian population-based Blue Mountains Eye Study (BMES). We replicated the association of I62V with estimated glomerular filtration rate (GFR; P=0.017) and creatinine clearance (CRCL; P=0.015). The minor allele of I62V (G) was deleterious: adding one copy of the G allele decreased GFR/CRCL by approximately 0.98 ml min(-1) per 1.73 m(2) (95% confidence interval (CI): 0.97, 0.99). We also replicated the association of Y402H with AMD and provided an unbiased estimate of population attributable risk (PAR). The minor allele of Y402H (C) was deleterious: the odds ratio estimate of CC genotype compared to TT was 1.87 (95% CI: 1.44, 2.45). The PAR of the C allele was estimated as 0.22 (95% CI: 0.15, 0.28). In summary, in the BMES population we confirmed the association between I62V and renal function, as measured by the estimated GFR, plus the association of Y402H with both early- and late-stage AMD.

Human molecular cytogenetics: diagnosis, prognosis, and disease management.

The year 2001 witnessed the sequencing of 90% of the euchromatic region in the human genome but the ultimate goal to delineate the positions of all genes is yet to be achieved. Fluorescence In Situ Hybridization (FISH) is one of the methods for localizing genes on chromosomes. In the present study, diagnostic utility of single-, dual-, and multicolor FISH was evaluated for prenatal diagnosis, cancer genetics, and screening of various congenital anomalies (sex chromosomal and autosomal). Centromeric probes for chromosomes X and Y were used for screening minor aneuploid cell lines (XXY, XO, and XXX) in the cases of primary amenorrhea and suspected Klinefelter syndrome. The cases with ambiguous genitalia were analyzed using a probe specific for the sex-determining region (SRY). Suspected cases of Down syndrome were subjected to FISH using probe specific for chromosome 21. FISH was also used to study gene alterations in retinoblastoma and myeloid leukemias. Prenatal diagnosis was done to screen for aneuploidies of chromosomes 13, 18, 21, X, and Y using FISH on uncultured cells from amniotic fluid and chorionic villi sampling. The screening for common aneuploidies was extended to abortuses from spontaneous abortions. Using FISH, low-level mosaicism could be identified in some cases of primary amenorrhea and suspected Klinefelter syndrome. Submicroscopic gene rearrangements could be detected using FISH in cases of ambiguous genitalia and cancers. Further interphase FISH could provide results within 24 hours. To conclude, FISH adds to the diagnostic utility of routine cytogenetics and its use on interphase nuclei overcomes the difficulty of conventional cytogenetics, thereby reducing the time between sampling and diagnosis to 24 hr.

Mutations in the Wolfram syndrome 1 gene (WFS1) are a common cause of low frequency sensorineural hearing loss.

Non-syndromic low frequency sensorineural hearing loss (LFSNHL) affecting only 2000 Hz and below is an unusual type of hearing loss that worsens over time without progressing to profound deafness. This type of LFSNHL may be associated with mild tinnitus but is not associated with vertigo. We have previously reported two families with autosomal dominant LFSNHL linked to adjacent but non-overlapping loci on 4p16, DFNA6 and DFNA14. However, further study revealed that an individual with LFSNHL in the DFNA6 family who had a recombination event that excluded the DFNA14 candidate region was actually a phenocopy, and consequently, DFNA6 and DFNA14 are allelic. LFSNHL appears to be genetically nearly homogeneous, as only one LFSNHL family is known to map to a different chromosome (DFNA1). The DFNA6/14 critical region includes WFS1, the gene responsible for Wolfram syndrome, an autosomal recessive disorder characterized by diabetes mellitus and optic atrophy, and often, deafness. Herein we report five different heterozygous missense mutations (T699M, A716T, V779M, L829P, G831D) in the WFS1 gene found in six LFSNHL families. Mutations in WFS1 were identified in all LFSNHL families tested, with A716T arising independently in two families. None of the mutations was found in at least 220 control chromosomes with the exception of V779M, which was identified in 1/336 controls. This frequency is consistent with the prevalence of heterozygous carriers for Wolfram syndrome estimated at 0.3-1%. An increased risk of sensorineural hearing loss has been reported in such carriers. Therefore, we conclude that mutations in WFS1 are a common cause of LFSNHL.

Parental age in Indian patients with sporadic hereditary retinoblastoma.

There is a consistent correlation between sporadic hereditary retinoblastoma and parental age. It has been proven beyond doubt that the birth rank is correlated with parental age. In the present study, a test for the effect of birth rank was performed in order to assess the risk of developing retinoblastoma with increased parental age. The study of the effect of birth rank showed a significant association between sporadic retinoblastoma (bilateral and unilateral) and late para, indicating that fresh germline mutations must have taken place in some of the sporadic cases. An investigation of the effect of birth rank on familial cases, obtained from published papers and our own series, showed that familial retinoblastoma is significantly associated with early para, suggesting early parental age. Further analysis of the mean paternal and maternal ages of sporadic cases (bilateral and unilateral) showed that the mean paternal age of sporadic bilateral (sporadic hereditary) cases was higher than that of sporadic unilateral cases (p<0.05). No such correlation was seen with mean maternal age. Thus, the present study shows that a high paternal age may be associated with sporadic bilateral (sporadic hereditary) retinoblastoma.

Nongonadal neoplasia in patients with Turner syndrome.

In a prospective study of 50 patients with retinoblastoma, a 10-year-old girl with unilateral (right eye) retinoblastoma was found to have 45,X karyotype. Because there is increasing evidence of nongonadal neoplasia occurring in patients with Turner syndrome in addition to the gonadal tumor from dysgenetic gonads, we reviewed the occurrence of nongonadal neoplasia in Turner syndrome cases. Of all the nongonadal neoplasia, neurogenic tumors show a preponderence among children and young adults with Turner syndrome. To the best of our knowledge, this is the first reported case of Turner syndrome with retinoblastoma. The available literature strongly suggests that patients with Turner syndrome may be at risk of developing neurogenic tumors. Further studies are necessary to identify the role of some X-linked genes that escape X-inactivation in tumorigenesis in patients with Turner syndrome.

Genetics of otosclerosis.

Otosclerosis is an early-middle adult life genetic disease affecting bone remodelling in the ear. Current knowledge of otosclerosis as an inherited disease dates to the mid-19th century, and we report here an attempt to understand the genetics of otosclerosis and detect its heterogeneity. The analysis was conducted on 151 otosclerotic families. The results of our study indicate that while heredity plays an important role in the manifestation of the disease a substantial portion of otosclerotic cases could arise due to non-genetic causes.

Absence of pericentromeric heterochromatin (9qh-) in a patient with bilateral retinoblastoma.

The polymorphisms of constitutive heterochromatin regions, present on chromosomes 1, 9, 16 and Y, are inherited in a Mendelian fashion. The C-band heteromorphism has been reported to be associated with various types of cancer. Heterochromatin is considered to play a role in protecting genome against the mutagens. Changes in the quantity and proportion of the different types of satellite DNA might increase the genetic susceptibility in people with heterochromatic variations, which in turn cause chromosome instability and predispose the individual to cancer. We report a case of bilateral retinoblastoma with complete absence of pericentromeric heterochromatin on one of the chromosomes number 9. A similar deficiency of pericentromeric heterochromatin on chromosome number 9 and 16 has been reported in a phenotypically normal individual and a Down syndrome case, respectively. This deficiency was found to be inherited from the father in all the three cases. Complete absence of pericentromeric heterochromatin of chromosome 9 is not being reported in association with cancer syndromes. Further studies are necessary to understand the role of this factor in normals and in those with cancer susceptibility, specially with retinoblastoma and the paternal origin of this deficiency.

Effects of inbreeding on reproductive losses in Kota tribe.

Sanghvi's hypothesis on long term effects of inbreeding was tested in Kotas. Kota is a numerically small tribal population in the Nilgiri district, Tamil Nadu State, India. Consanguineous marriages are common in this tribe. A total of 95 couples were taken for this study and necessary data were collected on a set proforma. Of the 95 couples, 28 (29.5%) were consanguineously related. The inbreeding coefficient for autosomal genes is 0.022 and for sex-linked genes is 0.03. Inbreeding effects on reproductive losses were examined through an exponential regression model. Although the regression coefficient B values are positive, they are insignificant, suggesting no consistent relationship between degree of consanguinity and the reproductive losses. The estimates of genetic load is 1.8 lethal equivalents per gamete and the average B/A ratio is 5. These findings empirically support the Sanghvi's contention.